Methods and reagents for detection of chikungunya virus and zika virus

ABSTRACT

Methods and oligonucleotide reagents for diagnosing chikungunya virus and Zika virus infections are described. In particular, the invention relates to quantitative assays that can detect all lineages of chikungunya virus and Zika virus and distinguish chikungunya virus and Zika virus from each other as well as dengue virus and other arbovirus pathogens.

TECHNICAL FIELD

The present invention pertains generally to chikungunya virus and Zikavirus and viral diagnostics for detecting these viruses individually orin combination. In particular, the invention relates to quantitativeassays that can detect all lineages of chikungunya virus and Zika virusand distinguish chikungunya virus and Zika virus from each other as wellas dengue virus and other arbovirus pathogens.

BACKGROUND

Over the past decade, chikungunya virus (CHIKV) has emerged from being arelatively rare arbovirus that caused sporadic outbreaks of humandisease in Africa and Asia to the cause of a pandemic that has affectedmillions of people across five continents (Powers (2010) Clin. Lab. Med.30:209-219). As a result, CHIKV has spread to new regions where denguevirus (DENV) is endemic, including, since December 2013, tropical andsubtropical regions of the Western Hemisphere (Leparc-Goffart et al.(2014) Lancet 383:514). This has created a new challenge for health caresystems that care for patients living in or returning from affectedareas, as the clinical presentation of dengue and chikungunya overlapsignificantly (Cleton et al. (2015) PLoS Negl Trop Dis 9:e0004073;Hochedez et al. (2008) Am. J. Trop. Med. Hyg. 78:710-713; Lee et al.(2012) PLoS Negl. Trop. Dis. 6:e1786; Mohd et al. (2013) J. Clin. Virol.56:141-145). The differentiation of infections with DENV and CHIKV isimportant not only for epidemiologic surveillance but also for clinicalcare, such as initiating appropriate management and providing prognosticinformation. While certain clinical and laboratory findings have beenassociated with dengue (thrombocytopenia, leukopenia) or chikungunya(arthralgia/arthritis), these are not sufficiently accurate to determinethe cause of illness in all cases without specific testing (Cleton etal., supra; Hochedez et al., supra; Mohd et al., supra).

Zika virus (ZIKV) is a mosquito-borne flavivirus that was firstidentified in a rhesus monkey from the Zika Forest of Uganda in 1947.Prior to 2007, very few cases of human infection with ZIKV had beenidentified and all had occurred in Africa or Asia. In 2007, a large ZIKVoutbreak occurred on Yap Island, Federated States of Micronesia.Patients presented with a rash, fever, arthralgias and conjunctivitis,and it was estimated that 73% of the Yap population older than 3 yearsof age was infected during this outbreak. The virus has since spread toislands of the South Pacific, and in May 2015, a large ZIKV outbreakbegan in northeastern Brazil. The virus has spread to at least sixstates in Brazil, with an estimate of 500,000-1,000,000 infections todate. Importantly, the virus has also been temporally linked to cases ofmicrocephaly in Brazil and neurologic malformations in babies bornduring outbreaks in the South Pacific.

ZIKV emergence in Brazil has coincided with an increase in cases of DENVinfection and ongoing CHIKV transmission. Human infection with ZIKVoften presents with symptoms that overlap with both of these viruses,which has created a new challenge for health care systems that care forpatients living in or returning from affected areas. The differentiationof infections with these viruses is important not only for epidemiologicsurveillance but also for clinical care, such as initiating appropriatemanagement and providing prognostic information. While certain clinicaland laboratory findings have been associated with dengue(thrombocytopenia, leukopenia) or chikungunya (arthralgia/arthritis),these are not sufficiently accurate to determine the cause of illness inall cases without specific testing. Clinical and laboratory findings inacute ZIKV infections have not been thoroughly studied owing to itsrecent emergence over the last few years.

Molecular tests are the most sensitive diagnostics for DENV, CHIKV, orZIKV in the acute setting. Serologic testing is also frequentlyperformed on paired acute and convalescent serum, but this can onlyconfirm a diagnosis in retrospect. Also, high rates of false-positiveDENV serologic results have been reported in patients with ZIKV. Anumber of molecular tests have been published for the detection of DENVand CHIKV, but only two real-time reverse transcriptase PCRs (rRT-PCRs)have been reported for ZIKV. Both of these assays are run as individualreactions. Molecular testing for all three viruses, using recommendedassays, can entail performing up to six amplification reactions for asingle patient sample followed by detection using gel electrophoresis.This results in increased costs, prolonged turn-around times, anddecreased rates of detection as all samples often cannot be tested foreach virus.

Thus, there remains a need for the development of effective strategiesfor the diagnosis, treatment, and prevention of chikungunya, Zika, anddengue viral infections. The availability of nucleic acid diagnostictests capable of efficiently detecting chikungunya, Zika, and dengueviruses in human specimens such as plasma, serum and respiratorysecretions will assist the medical community in better diagnosing andtreating these arbovirus infections.

SUMMARY

The present invention is based on the development of sensitive, reliablenucleic acid-based diagnostic assays for the detection of chikungunyaand Zika viruses in biological samples from potentially infectedsubjects. The assays allow rapid detection of all lineages ofchikungunya virus and Zika virus and can distinguish chikungunya virusand Zika virus from each other as well as from other arboviruspathogens. The methods can also be used to quantitate the amount ofvirus that is present in a biological sample. If infection is detected,an individual can be given appropriate therapy, and steps can be takento prevent or reduce further transmission and spread of the viruses. Ifinfection is ruled-out, other potential causes of undifferentiatedfebrile illness can be further investigated.

In addition, the assays described herein can be readily combined withother assays for detection of other arbovirus pathogens. Multiplexassays can be used to detect infection by a single virus or coinfectionby more than one virus. In particular, multiplex assays can be used todetect chikungunya virus, Zika virus, and dengue virus, or anycombination thereof in a single assay to determine if an individual isinfected with any of these viruses or coinfected with more than onevirus.

The methods utilize primers and probes for amplifying and/or detectingtarget sequences of one or more chikungunya virus, Zika virus, or denguevirus genotypes, to allow detection of a single viral genotype ormultiple genotypes simultaneously in a single assay. In certainembodiments, the virus sequences are detected using reversetranscriptase-polymerase chain reaction (RT-PCR), for example, usingreal-time RT-PCR and/or multiplex RT-PCR. Other nucleic-acid baseddetection techniques such as, but not limited to, nucleic acid sequencebased amplification (NASBA), a 5′ nuclease assay (e.g., TaqMan), ortranscription-mediated amplification (TMA), can also be used.

Exemplary primers (SEQ ID NO:6 and SEQ ID NO:7) and probes (SEQ ID NO:8)for detection of chikungunya virus are shown in Example 1 (see Table 1).Changes to the nucleotide sequences of these primers and probes may beintroduced corresponding to genetic variations in particular chikungunyastrains. For example up to three nucleotide changes, including 1nucleotide change, 2 nucleotide changes, or three nucleotide changes,may be made in a sequence selected from the group consisting of SEQ IDNOS:6-8, wherein the oligonucleotide primer or probe is capable ofhybridizing to and amplifying or detecting a particular chikungunyavirus target nucleic acid (e.g., a portion of an NSP2 gene).

Exemplary primers (SEQ ID NOS:9-16) and probes (SEQ ID NOS:17-25) fordetection of dengue virus in combination with chikungunya virus and/orZika virus in multiplex assays are also shown in Example 1 (see Tables 4and 5). Changes to the nucleotide sequences of these primers and probesmay be introduced corresponding to genetic variations in particulardengue strains. For example up to three nucleotide changes, including 1nucleotide change, 2 nucleotide changes, or three nucleotide changes,may be made in a sequence selected from the group consisting of SEQ IDNOS:9-25, wherein the oligonucleotide primer or probe is capable ofhybridizing to and amplifying or detecting a particular dengue virustarget nucleic acid.

In one aspect, the invention includes a composition for detectingchikungunya virus in a biological sample using a nucleic acidamplification assay, the composition comprising at least one set ofoligonucleotide primers comprising a forward primer and a reverse primercapable of amplifying at least a portion of a chikungunya virus genome,wherein the primers are not more than 40 nucleotides in length, whereinthe set of primers is selected from the group consisting of: a) aforward primer comprising the nucleotide sequence of SEQ ID NO:6 and areverse primer comprising the sequence of SEQ ID NO:7; b) a forwardprimer comprising at least 10 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:6 and a reverse primer comprising at least 10contiguous nucleotides of the nucleotide sequence of SEQ ID NO:7; c) aforward primer comprising a nucleotide sequence having at least 95%identity to the sequence of SEQ ID NO:6 and a reverse primer comprisinga nucleotide sequence having at least 95% identity to the sequence ofSEQ ID NO:7, wherein the primer is capable of hybridizing to andamplifying chikungunya virus nucleic acids in the nucleic acidamplification assay; d) a forward primer and a reverse primer comprisingat least one nucleotide sequence that differs from the correspondingnucleotide sequence of the forward primer or reverse primer of theprimer set of (a) in that the primer has up to three nucleotide changescompared to the corresponding sequence, wherein the primer is capable ofhybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; and e) a forward primer and a reverseprimer comprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(d).

In certain embodiments, the composition may further comprise at leastone detectably labeled oligonucleotide probe sufficiently complementaryto and capable of hybridizing with a chikungunya virus RNA or anamplicon thereof. An exemplary probe comprises the nucleotide sequenceof SEQ ID NO:8. The composition may include a set of probes capable ofdetecting multiple genotypes of chikungunya virus, including any viralstrain of any of the chikungunya virus genotypes (e.g., West African,East/Central/South African (ECSA), and Asian (Indian and south eastclades)). In one embodiment, the probe is selected from the groupconsisting of: a) a probe comprising the sequence of SEQ ID NO:8; b) aprobe comprising a nucleotide sequence having at least 95% identity tothe sequence of SEQ ID NO:8, wherein the probe is capable of hybridizingto and detecting the chikungunya virus RNA or an amplicon thereof; andc) a probe that differs from the corresponding nucleotide sequence ofSEQ ID NO:8 by up to three nucleotide changes, wherein the probe iscapable of hybridizing to and detecting the chikungunya virus RNA or anamplicon thereof.

The probe may be detectably labeled with a fluorophore (e.g., afluorescein or rhodamine derivative). The fluorophore may include, butis not limited to, a CAL Fluor dye, a Quasar dye, an Alexa Fluor, 2′,4′, 5′, 7′-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein(FAM), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635.

In certain embodiments, the probe comprises a 5′-fluorophore and a3′-quencher. The 3′-quencher may include, but is not limited to, a blackhole quencher (e.g., BHQ-0, BHQ-1, BHQ-2, and BHQ-3), tetramethylrhodamine (TAMRA), dabcyl, a Qxl quencher, an Iowa black quencher, anEclipse quencher, an ATTO quencher, and dihydrocyclopyrroloindoletripeptide minor groove binder (MGB). In one embodiment, the probe is amolecular beacon.

In one embodiment, the composition comprises a primer comprising thesequence of SEQ ID NO:6, a primer comprising the sequence of SEQ IDNO:7, and a probe comprising the sequence of SEQ ID NO:8.

In another aspect, the invention includes a method for detectingchikungunya virus, the method comprising: a) contacting nucleic acids ofa biological sample suspected of containing chikungunya virus with acomposition, as described herein, for detecting chikungunya virus bynucleic acid amplification of viral RNA, b) amplifying at least aportion of a chikungunya virus RNA, if present, wherein the chikungunyavirus RNA comprises an NSP2 target sequence; and c) detecting thepresence of the amplified nucleic acids using at least one detectablylabeled oligonucleotide probe sufficiently complementary to and capableof hybridizing with the chikungunya virus RNA or amplicon thereof, ifpresent, as an indication of the presence or absence of chikungunyavirus in the sample. In one embodiment, the method further comprisesisolating the chikungunya virus nucleic acids from the biological sampleprior to amplification. In another embodiment, the method furthercomprises isolating the chikungunya virus nucleic acids from thebiological sample after amplification.

In another embodiment, the method is performed with at least one probeselected from the group consisting of a) a probe comprising the sequenceof SEQ ID NO:8; a probe comprising a nucleotide sequence having at least95% identity to the sequence of SEQ ID NO:8, wherein the probe iscapable of hybridizing to and detecting the chikungunya virus RNA or anamplicon thereof; and a probe that differs from the correspondingnucleotide sequence of SEQ ID NO:8 by up to three nucleotide changes,wherein the probe is capable of hybridizing to and detecting thechikungunya virus RNA or an amplicon thereof.

In certain embodiments, the probe used in the method of detectingchikungunya virus comprises a fluorophore (e.g., fluorescein orrhodamine derivative). The fluorophore may include, but is not limitedto, a CAL Fluor dye, a Quasar dye, an Alexa Fluor, 2′, 4′, 5′,7′-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635.

In certain embodiments, the detectably labeled probe comprises a5′-fluorophore and a 3′-quencher. The 3′-quencher may include, but isnot limited to, a black hole quencher (e.g., BHQ-0, BHQ-1, BHQ-2, andBHQ-3), tetramethyl rhodamine (TAMRA), dabcyl, a Qxl quencher, an Iowablack quencher, an Eclipse quencher, an ATTO quencher, anddihydrocyclopyrroloindole tripeptide minor groove binder (MGB). In oneembodiment, the probe used for detection of chikungunya virus is amolecular beacon.

In another embodiment, the set of primers and probes that are used fordetecting chikungunya virus in the biological sample comprise a primercomprising the sequence of SEQ ID NO:6, a primer comprising the sequenceof SEQ ID NO:7, and a probe comprising the sequence of SEQ ID NO:8.

In certain embodiments, the method further comprises detecting denguevirus in the biological sample. In one embodiment, the primers used fordetecting dengue virus in the biological sample comprise a primercomprising the sequence of SEQ ID NO:9, a primer comprising the sequenceof SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, aprimer comprising the sequence of SEQ ID NO:12, a primer comprising thesequence of SEQ ID NO:13, a primer comprising the sequence of SEQ IDNO:14, a primer comprising the sequence of SEQ ID NO:15, and a primercomprising the sequence of SEQ ID NO:16.

In another embodiment, the method further comprises using at least oneprobe for detecting dengue virus, wherein at least one probe is selectedfrom the group consisting of: a) a probe comprising the sequence of SEQID NO:17, b) a probe comprising the sequence of SEQ ID NO:18, c) a probecomprising the sequence of SEQ ID NO:19, d) a probe comprising thesequence of SEQ ID NO:20, e) a probe comprising the sequence of SEQ IDNO:21, f) a probe comprising the sequence of SEQ ID NO:22, g) a probecomprising the sequence of SEQ ID NO:23, h) a probe comprising thesequence of SEQ ID NO:24, i) a probe comprising the sequence of SEQ IDNO:25, and j) a probe that differs from the corresponding nucleotidesequence of a probe selected from the group consisting of (a)-(i) inthat the probe has up to three nucleotide changes compared to thecorresponding sequence, wherein the probe is capable of hybridizing toand detecting the dengue virus RNA or amplicon thereof.

In another embodiment, a set of probes is used for detecting denguevirus in a biological sample, wherein the set of probes comprises aprobe comprising the sequence of SEQ ID NO:17, a probe comprising thesequence of SEQ ID NO:18, a probe comprising the sequence of SEQ IDNO:19, and a probe comprising the sequence of SEQ ID NO:20.

In another embodiment, a set of probes is used for detecting denguevirus in a biological sample, wherein the set of probes comprises aprobe comprising the sequence of SEQ ID NO:22, a probe comprising thesequence of SEQ ID NO:23, a probe comprising the sequence of SEQ IDNO:24, and a probe comprising the sequence of SEQ ID NO:25.

In another embodiment a set of primers and probes are used for detectingdengue virus in a biological sample comprising: a primer comprising thesequence of SEQ ID NO:9, a primer comprising the sequence of SEQ IDNO:10, a primer comprising the sequence of SEQ ID NO:11, a primercomprising the sequence of SEQ ID NO:12, a primer comprising thesequence of SEQ ID NO:13, a primer comprising the sequence of SEQ IDNO:14, a primer comprising the sequence of SEQ ID NO:15, a primercomprising the sequence of SEQ ID NO:16, a probe comprising the sequenceof SEQ ID NO:17, a probe comprising the sequence of SEQ ID NO:18, aprobe comprising the sequence of SEQ ID NO:19, and a probe comprisingthe sequence of SEQ ID NO:20. In one embodiment, the probe comprisingthe sequence of SEQ ID NO:17 further comprises a 5′ FAM fluorophore anda 3′ BHQ-1 quencher, the probe comprising the sequence of SEQ ID NO:18further comprises a 5′ CAL Fluor Orange 560 fluorophore and a 3′ BHQ-1quencher, the probe comprising the sequence of SEQ ID NO:19 furthercomprises a 5′ CAL Fluor Red 610 fluorophore and a 3′ BHQ-2 quencher,and the probe comprising the sequence of SEQ ID NO:20 further comprisesa 5′ Quasar Blue 670 fluorophore and a 3′ BHQ-2 quencher.

In another embodiment, a set of primers and probes are used fordetecting dengue virus in a biological sample comprising: a primercomprising the sequence of SEQ ID NO:9, a primer comprising the sequenceof SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, aprimer comprising the sequence of SEQ ID NO:12, a primer comprising thesequence of SEQ ID NO:13, a primer comprising the sequence of SEQ IDNO:14, a primer comprising the sequence of SEQ ID NO:15, a primercomprising the sequence of SEQ ID NO:16, a probe comprising the sequenceof SEQ ID NO:22, a probe comprising the sequence of SEQ ID NO:23, aprobe comprising the sequence of SEQ ID NO:24, and a probe comprisingthe sequence of SEQ ID NO:25. In one embodiment, the probe comprisingthe sequence of SEQ ID NO:22 further comprises a 5′ FAM fluorophore anda 3′ BHQplus quencher, the probe comprising the sequence of SEQ ID NO:23further comprises a 5′ FAM fluorophore and a 3′ BHQplus quencher, theprobe comprising the sequence of SEQ ID NO:24 further comprises a 5′ FAMfluorophore and a 3′ BHQplus quencher, and the probe comprising thesequence of SEQ ID NO:25 further comprises a FAM fluorophore and a 3′BHQplus quencher.

In certain embodiments, the method further comprises distinguishingchikungunya virus nucleic acids from dengue virus, West Nile virus,Japanese encephalitis virus, tick-borne encephalitis virus, yellow fevervirus, Saint Louis encephalitis virus, Zika virus, o'nyong-nyong virus,Semliki Forest virus, mayaro virus, Ross River virus, Getah virus,Barmah Forest virus, Una virus, hepatitis C virus (HCV), humanimmunodeficiency virus (HIV), Leptospira, Plasmodium species, or RiftValley fever virus nucleic acids.

In another aspect, the invention includes an isolated oligonucleotidenot more than 40 nucleotides in length comprising: a) a nucleotidesequence comprising at least 10 contiguous nucleotides from a nucleotidesequence selected from the group consisting of SEQ ID NOS:6-8; b) anucleotide sequence having at least 95% identity to a nucleotidesequence selected from the group consisting of SEQ ID NO:6-8, whereinthe oligonucleotide is capable of hybridizing to and amplifying ordetecting a chikungunya virus nucleic acid; c) a nucleotide sequencethat differs from a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:6-8 by up to three nucleotide changes, whereinthe oligonucleotide is capable of hybridizing to and amplifying ordetecting a chikungunya virus nucleic acid; or d) complements of(a)-(c). Oligonucleotides may further comprise a detectable label. Forexample, the detectable label may be a fluorophore (e.g., fluorescein orrhodamine derivative). The fluorophore may include, but is not limitedto, a CAL Fluor dye, a Quasar dye, an Alexa Fluor, 2′, 4′, 5′,7′-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635. The oligonucleotide may further comprise aquencher such as, but not limited to black hole quencher (e.g., BHQ-0,BHQ-1, BHQ-2, and BHQ-3), tetramethyl rhodamine (TAMRA), dabcyl, a Qxlquencher, an Iowa black quencher, an Eclipse quencher, an ATTO quencher,and dihydrocyclopyrroloindole tripeptide minor groove binder (MGB). Incertain embodiments, the oligonucleotide comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOS:6-8 or a complementthereof.

In another aspect, the invention includes a kit for detectingchikungunya virus in a biological sample by nucleic acid amplificationof viral RNA. The kit may comprise a composition, as described herein,comprising at least one set of primers including a forward primer and areverse primer capable of amplifying at least a portion of a chikungunyavirus genome, including an NSP2 target sequence. The kit may furthercomprise written instructions for identifying the presence of thechikungunya virus, quantitating the chikungunya virus, and/or serotypingthe chikungunya virus. The kit may also comprise reagents for performingreverse transcriptase polymerase chain reaction (RT-PCR), nucleic acidsequence based amplification (NASBA), transcription-mediatedamplification (TMA), a fluorogenic 5′ nuclease assay, or other nucleicacid amplification technique. In one embodiment, the kit furthercomprises oligonucleotide primers and probes for detecting Zika virusand/or dengue virus as described herein.

The methods of the invention can be used to detect chikungunya virus inbiological samples such as, but not limited to, blood, plasma, serum,saliva, cerebrospinal fluid (CSF), fibroblasts, epithelial cells,endothelial cells, macrophages, skin, liver, muscle, spleen, lymphnodes, thymus, lung, kidneys, or bone marrow. Chikungunya virus can bespecifically detected even in samples containing other viruses orpathogens, such as West Nile virus, Japanese encephalitis virus,tick-borne encephalitis virus, yellow fever virus, Saint Louisencephalitis virus, Zika virus, o'nyong-nyong virus, Semliki Forestvirus, mayaro virus, Ross River virus, Getah virus, Barmah Forest virus,Una virus, hepatitis C virus (HCV), Leptospira, Plasmodium species, orRift Valley fever virus. Moreover, the assays can be used to screenmosquitoes, monkeys, birds, cattle, rodents, and other hosts forchikungunya virus in order to determine if a particular insect or animalpopulation is infected with the virus, thereby preventing furthertransmission and spread of chikungunya virus infection. Individualscoinfected with chikungunya virus and other arbovirus pathogens (e.g.,dengue virus) can also be identified using multiplex assays.Additionally, infected blood samples can be detected and excluded fromtransfusion, as well as from the preparation of blood derivatives.

Exemplary primers (SEQ ID NOS:27-29) and probes (SEQ ID NO:30) fordetection of Zika virus are shown in Example 2 (see Table 6). Changes tothe nucleotide sequences of these primers and probes may be introducedcorresponding to genetic variations in particular Zika virus strains.For example up to three nucleotide changes, including 1 nucleotidechange, 2 nucleotide changes, or three nucleotide changes, may be madein a sequence selected from the group consisting of SEQ ID NOS:27-30,wherein the oligonucleotide primer or probe is capable of hybridizing toand amplifying or detecting a particular Zika virus target nucleic acid(e.g., a conserved viral sequence).

In another aspect, the invention includes a composition for detectingZika virus in a biological sample using a nucleic acid amplificationassay, the composition comprising at least one set of oligonucleotideprimers comprising a forward primer and at least one reverse primercapable of amplifying at least a portion of a Zika virus genome, whereinthe primers are not more than 40 nucleotides in length, wherein the setof primers is selected from the group consisting of: a) a forward primercomprising the nucleotide sequence of SEQ ID NO:27 and a reverse primercomprising the sequence of SEQ ID NO:28; b) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:27 and a reverse primer comprisingthe sequence of SEQ ID NO:29; c) a forward primer comprising thenucleotide sequence of SEQ ID NO:27, a first reverse primer comprisingthe sequence of SEQ ID NO:28, and a second reverse primer comprising thesequence of SEQ ID NO:29; d) a forward primer comprising at least 10contiguous nucleotides of the nucleotide sequence of SEQ ID NO:27 and atleast one reverse primer comprising at least 10 contiguous nucleotidesof a nucleotide sequence selected from the group consisting of SEQ IDNO:28 and SEQ ID NO:29; e) a forward primer comprising a nucleotidesequence having at least 95% identity to the sequence of SEQ ID NO:27and at least one reverse primer comprising a nucleotide sequence havingat least 95% identity to a sequence selected from the group consistingof SEQ ID NO:28 and SEQ ID NO:29, wherein the primer is capable ofhybridizing to and amplifying Zika virus nucleic acids in the nucleicacid amplification assay; f) a forward primer and a reverse primercomprising at least one nucleotide sequence that differs from thecorresponding nucleotide sequence of the forward primer or reverseprimer of the primer set of (a) or (b) in that the primer has up tothree nucleotide changes compared to the corresponding sequence, whereinthe primer is capable of hybridizing to and amplifying Zika virusnucleic acids in the nucleic acid amplification assay; and g) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(f).

In certain embodiments, the composition may further comprise at leastone detectably labeled oligonucleotide probe sufficiently complementaryto and capable of hybridizing with a Zika virus RNA or an ampliconthereof. An exemplary probe comprises the nucleotide sequence of SEQ IDNO:30. The composition may include a set of probes capable of detectingmultiple genotypes of Zika virus, including any viral strain of any ofthe Zika virus genotypes. In certain embodiments, the probe is selectedfrom the group consisting of: a) a probe comprising the sequence of SEQID NO:30; b) a probe comprising a nucleotide sequence having at least95% identity to the sequence of SEQ ID NO:30, wherein the probe iscapable of hybridizing to and detecting the Zika virus RNA or anamplicon thereof; and c) a probe that differs from the correspondingnucleotide sequence of SEQ ID NO:30 by up to three nucleotide changes,wherein the probe is capable of hybridizing to and detecting the Zikavirus RNA or an amplicon thereof.

In another embodiment, the composition comprises a primer comprising thesequence of SEQ ID NO:27, a primer comprising the sequence of SEQ IDNO:28, a primer comprising the sequence of SEQ ID NO:29, and a probecomprising the sequence of SEQ ID NO:30.

The probe may be detectably labeled with a fluorophore (e.g., afluorescein or rhodamine derivative). The fluorophore may include, butis not limited to, a CAL Fluor dye, a Quasar dye, an Alexa Fluor, 2′,4′, 5′, 7′-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein(FAM), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635.

In certain embodiments, the probe comprises a 5′-fluorophore and a3′-quencher. The 3′-quencher may include, but is not limited to, a blackhole quencher (e.g., BHQ-0, BHQ-1, BHQ-2, and BHQ-3), tetramethylrhodamine (TAMRA), dabcyl, a Qxl quencher, an Iowa black quencher, anEclipse quencher, an ATTO quencher, and dihydrocyclopyrroloindoletripeptide minor groove binder (MGB). In one embodiment, the probe is amolecular beacon.

In another aspect, the invention includes a method for detecting Zikavirus, the method comprising: a) contacting nucleic acids of abiological sample suspected of containing Zika virus with a composition,as described herein, for detecting Zika virus by nucleic acidamplification of viral RNA, b) amplifying at least a portion of a Zikavirus RNA, if present; and c) detecting the presence of the amplifiednucleic acids using at least one detectably labeled oligonucleotideprobe sufficiently complementary to and capable of hybridizing with theZika virus RNA or amplicon thereof, if present, as an indication of thepresence or absence of Zika virus in the sample. In one embodiment, themethod further comprises isolating the Zika virus nucleic acids from thebiological sample prior to amplification. In another embodiment, themethod further comprises isolating the Zika virus nucleic acids from thebiological sample after amplification.

In another embodiment, the method is performed with at least one probeselected from the group consisting of a) a probe comprising the sequenceof SEQ ID NO:30; a probe comprising a nucleotide sequence having atleast 95% identity to the sequence of SEQ ID NO:30, wherein the probe iscapable of hybridizing to and detecting the Zika virus RNA or anamplicon thereof; and a probe that differs from the correspondingnucleotide sequence of SEQ ID NO:30 by up to three nucleotide changes,wherein the probe is capable of hybridizing to and detecting the Zikavirus RNA or an amplicon thereof.

In certain embodiments, the probe used in the method of detecting Zikavirus comprises a fluorophore (e.g., fluorescein or rhodaminederivative). The fluorophore may include, but is not limited to, a CALFluor dye, a Quasar dye, an Alexa Fluor, 2′, 4′, 5′,7′-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635.

In certain embodiments, the detectably labeled probe comprises a5′-fluorophore and a 3′-quencher. The 3′-quencher may include, but isnot limited to, a black hole quencher (e.g., BHQ-0, BHQ-1, BHQ-2, andBHQ-3), tetramethyl rhodamine (TAMRA), dabcyl, a Qxl quencher, an Iowablack quencher, an Eclipse quencher, an ATTO quencher, anddihydrocyclopyrroloindole tripeptide minor groove binder (MGB). In oneembodiment, the probe used for detection of Zika virus is a molecularbeacon.

In another embodiment, the set of primers and probes that are used fordetecting Zika virus in the biological sample comprise a primercomprising the sequence of SEQ ID NO:27, a primer comprising thesequence of SEQ ID NO:28, a primer comprising the sequence of SEQ IDNO:29, and a probe comprising the sequence of SEQ ID NO:30.

In certain embodiments, the method further comprises distinguishing Zikavirus nucleic acids from dengue virus, chikungunya virus, West Nilevirus, Japanese encephalitis virus, tick-borne encephalitis virus,yellow fever virus, Saint Louis encephalitis virus, o'nyong-nyong virus,Semliki Forest virus, mayaro virus, Ross River virus, Getah virus,Barmah Forest virus, Una virus, hepatitis C virus (HCV), humanimmunodeficiency virus (HIV), Leptospira, Plasmodium species, or RiftValley fever virus nucleic acids.

In certain embodiments, the method further comprises detecting denguevirus and/or chikungunya virus in the biological sample. In oneembodiment, detecting the chikungunya virus comprises amplifying atleast a portion of the chikungunya virus RNA using at least one set ofprimers comprising a forward primer and a reverse primer capable ofamplifying at least a portion of a chikungunya virus genome comprisingan NSP2 target sequence, wherein the primers are not more than about 40nucleotides in length, wherein the set of primers is selected from thegroup consisting of: a) a forward primer comprising the nucleotidesequence of SEQ ID NO:6 and a reverse primer comprising the sequence ofSEQ ID NO:7; b) a forward primer comprising at least 10 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:6 and a reverseprimer comprising at least 10 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:7; c) a forward primer comprising a nucleotidesequence having at least 95% identity to the sequence of SEQ ID NO:6 anda reverse primer comprising a nucleotide sequence having at least 95%identity to the sequence of SEQ ID NO:7, wherein the primer is capableof hybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; d) a forward primer and a reverseprimer comprising at least one nucleotide sequence that differs from thecorresponding nucleotide sequence of the forward primer or reverseprimer of the primer set of (a) in that the primer has up to threenucleotide changes compared to the corresponding sequence, wherein theprimer is capable of hybridizing to and amplifying chikungunya virusnucleic acids in the nucleic acid amplification assay; and e) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(d).

The amplified chikungunya virus nucleic acids can be detected using atleast one detectably labeled oligonucleotide probe sufficientlycomplementary to and capable of hybridizing with the chikungunya virusRNA or amplicon thereof, if present, as an indication of the presence orabsence of chikungunya virus in the sample. In certain embodiments, atleast one probe is selected from the group consisting of: a) a probecomprising the sequence of SEQ ID NO:8; b) a probe comprising anucleotide sequence having at least 95% identity to the sequence of SEQID NO:8, wherein the probe is capable of hybridizing to and detectingthe chikungunya virus RNA or an amplicon thereof; and c) a probe thatdiffers from the corresponding nucleotide sequence of SEQ ID NO:8 by upto three nucleotide changes, wherein the probe is capable of hybridizingto and detecting the chikungunya virus RNA or an amplicon thereof.

In another embodiment, detecting the chikungunya virus comprisesamplifying at least a portion of the chikungunya virus RNA using atleast one set of primers comprising a forward primer and a reverseprimer capable of amplifying at least a portion of a chikungunya virusgenome comprising an NSP2 target sequence, wherein the primers are notmore than about 40 nucleotides in length, wherein the set of primers isselected from the group consisting of: a) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:1 and at least one reverse primercomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:3; b) a forward primer comprising at least 10contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1 and atleast one reverse primer comprising at least 10 contiguous nucleotidesof a nucleotide sequence selected from the group consisting of SEQ IDNO:2 and SEQ ID NO:3; c) a forward primer comprising a nucleotidesequence having at least 95% identity to the sequence of SEQ ID NO:1 andat least one reverse primer comprising a nucleotide sequence having atleast 95% identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3, wherein the primer is capableof hybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; d) a forward primer and a reverseprimer comprising at least one nucleotide sequence that differs from thecorresponding nucleotide sequence of the forward primer or reverseprimer of the primer set of (a) in that the primer has up to threenucleotide changes compared to the corresponding sequence, wherein theprimer is capable of hybridizing to and amplifying chikungunya virusnucleic acids in the nucleic acid amplification assay; and e) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(d). The amplified nucleic acids can be detected usingat least one detectably labeled oligonucleotide probe sufficientlycomplementary to and capable of hybridizing with the chikungunya virusRNA or amplicon thereof, if present, as an indication of the presence orabsence of chikungunya virus in the sample. In certain embodiments, atleast one probe is selected from the group consisting of: a) a probecomprising a sequence selected from the group consisting of SEQ ID NO:4and SEQ ID NO:5; b) a probe comprising a nucleotide sequence having atleast 95% identity to a sequence selected from the group consisting ofSEQ ID NO:4 and SEQ ID NO:5, wherein the probe is capable of hybridizingto and detecting the chikungunya virus RNA or an amplicon thereof; andc) a probe that differs from the corresponding nucleotide sequence ofSEQ ID NO:4 or SEQ ID NO:5 by up to three nucleotide changes, whereinthe probe is capable of hybridizing to and detecting the chikungunyavirus RNA or an amplicon thereof.

In certain embodiments, the method further comprises detecting denguevirus in the biological sample. In one embodiment, the primers used fordetecting dengue virus in the biological sample comprise a primercomprising the sequence of SEQ ID NO:9, a primer comprising the sequenceof SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, aprimer comprising the sequence of SEQ ID NO:12, a primer comprising thesequence of SEQ ID NO:13, a primer comprising the sequence of SEQ IDNO:14, a primer comprising the sequence of SEQ ID NO:15, and a primercomprising the sequence of SEQ ID NO:16.

In another embodiment, the method further comprises using at least oneprobe for detecting dengue virus, wherein at least one probe is selectedfrom the group consisting of: a) a probe comprising the sequence of SEQID NO:17, b) a probe comprising the sequence of SEQ ID NO:18, c) a probecomprising the sequence of SEQ ID NO:19, d) a probe comprising thesequence of SEQ ID NO:20, e) a probe comprising the sequence of SEQ IDNO:21, f) a probe comprising the sequence of SEQ ID NO:22, g) a probecomprising the sequence of SEQ ID NO:23, h) a probe comprising thesequence of SEQ ID NO:24, i) a probe comprising the sequence of SEQ IDNO:25, and j) a probe that differs from the corresponding nucleotidesequence of a probe selected from the group consisting of (a)-(i) inthat the probe has up to three nucleotide changes compared to thecorresponding sequence, wherein the probe is capable of hybridizing toand detecting the dengue virus RNA or amplicon thereof.

In another aspect, the invention includes an isolated oligonucleotidenot more than 40 nucleotides in length comprising: a) a nucleotidesequence comprising at least 10 contiguous nucleotides from a nucleotidesequence selected from the group consisting of SEQ ID NOS:27-30; b) anucleotide sequence having at least 95% identity to a nucleotidesequence selected from the group consisting of SEQ ID NOS:27-30, whereinthe oligonucleotide is capable of hybridizing to and/or amplifying Zikavirus nucleic acids; c) a nucleotide sequence that differs from anucleotide sequence selected from the group consisting of SEQ IDNOS:27-30 by up to three nucleotide changes, wherein the oligonucleotideis capable of hybridizing to and/or amplifying Zika virus nucleic acids;or d) complements of (a)-(c). Oligonucleotides may further comprise adetectable label. For example, the detectable label may be a fluorophore(e.g., fluorescein or rhodamine derivative). The fluorophore mayinclude, but is not limited to, a CAL Fluor dye, a Quasar dye, an AlexaFluor, 2′, 4′, 5′, 7′-tetrachloro-4-7-dichlorofluorescein (TET),carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), Cy3, Cy5, and Texas Red. In one embodiment, thefluorophore is a CAL Fluor dye selected from the group consisting of CALFluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red610, and CAL Fluor Red 635. The oligonucleotide may further comprise aquencher such as, but not limited to black hole quencher (e.g., BHQ-0,BHQ-1, BHQ-2, and BHQ-3), tetramethyl rhodamine (TAMRA), dabcyl, a Qxlquencher, an Iowa black quencher, an Eclipse quencher, an ATTO quencher,and dihydrocyclopyrroloindole tripeptide minor groove binder (MGB). Incertain embodiments, the oligonucleotide comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOS:27-30 or a complementthereof.

In another aspect, the invention includes a kit for detecting Zika virusin a biological sample by nucleic acid amplification of viral RNA. Thekit may comprise a composition, as described herein, comprising at leastone set of primers including a forward primer and a reverse primercapable of amplifying at least a portion of a Zika virus genome. The kitmay further comprise written instructions for identifying the presenceof the Zika virus, quantitating the Zika virus, and/or serotyping theZika virus. The kit may also comprise reagents for performing reversetranscriptase polymerase chain reaction (RT-PCR), nucleic acid sequencebased amplification (NASBA), transcription-mediated amplification (TMA),a fluorogenic 5′ nuclease assay, or other nucleic acid amplificationtechnique. In one embodiment, the kit further comprises oligonucleotideprimers and probes for detecting dengue virus and/or chikungunya virus.

The methods of the invention can be used to detect Zika virus inbiological samples such as, but not limited to, blood, plasma, serum,saliva, cerebrospinal fluid (CSF), urine, amniotic fluid, dendriticcells, fibroblasts, keratinocytes, epithelial cells, endothelial cells,macrophages, and tissue samples obtained from the skin, liver, muscles,joints, spleen, lymph nodes, thymus, lung, brain, nerves, kidneys, orbone marrow. Zika virus can be specifically detected even in samplescontaining other viruses or pathogens, such as dengue virus, chikungunyavirus, West Nile virus, Japanese encephalitis virus, tick-borneencephalitis virus, yellow fever virus, Saint Louis encephalitis virus,o'nyong-nyong virus, Semliki Forest virus, mayaro virus, Ross Rivervirus, Getah virus, Barmah Forest virus, Una virus, hepatitis C virus(HCV), Leptospira, Plasmodium species, or Rift Valley fever virus.Moreover, the assays can be used to screen mosquitoes, monkeys, birds,cattle, rodents, and other hosts for Zika virus in order to determine ifa particular insect or animal population is infected with the virus,thereby preventing further transmission and spread of Zika virusinfection. Individuals coinfected with Zika virus and other arboviruspathogens (e.g., dengue virus or chikungunya virus) can also beidentified using multiplex assays. Additionally, infected blood samplescan be detected and excluded from transfusion, as well as from thepreparation of blood derivatives.

In certain embodiments, the kit comprises: written instructions foridentifying the presence of Zika virus; and at least one set of primerscomprising a forward primer and a reverse primer capable of amplifyingat least a portion of a Zika virus genome, wherein the primers are notmore than about 40 nucleotides in length, wherein the set of primers isselected from the group consisting of: a) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:27 and at least one reverse primercomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:28 and SEQ ID NO:29; b) a forward primer comprising at least10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:27 andat least one reverse primer comprising at least 10 contiguousnucleotides of a nucleotide sequence selected from the group consistingof SEQ ID NO:28 and SEQ ID NO:29; c) a forward primer comprising anucleotide sequence having at least 95% identity to the sequence of SEQID NO:27 and at least one reverse primer comprising a nucleotidesequence having at least 95% identity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:28 and SEQ ID NO:29, wherein theprimer is capable of hybridizing to and amplifying Zika virus nucleicacids in the nucleic acid amplification assay; d) a forward primer andat least one reverse primer comprising at least one nucleotide sequencethat differs from the corresponding nucleotide sequence of the forwardprimer or at least one reverse primer of the primer set of (a) in thatthe primer has up to three nucleotide changes compared to thecorresponding sequence, wherein the primer is capable of hybridizing toand amplifying Zika virus nucleic acids in the nucleic acidamplification assay; and e) a forward primer and at least one reverseprimer comprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(d). Inone embodiment, the kit comprises a forward primer comprising thenucleotide sequence of SEQ ID NO:27, a first reverse primer comprisingthe nucleotide sequence of SEQ ID NO:28, and a second reverse primercomprising the nucleotide sequence of SEQ ID NO:29.

Additionally, the kit may further comprises at least one probe fordetecting Zika virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:30; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:30, whereinthe probe is capable of hybridizing to and detecting the Zika virus RNAor an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:30 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the Zika virus RNA or an amplicon thereof. In one embodiment,the kit comprises a forward primer comprising the nucleotide sequence ofSEQ ID NO:27, a first reverse primer comprising the nucleotide sequenceof SEQ ID NO:28, a second reverse primer comprising the nucleotidesequence of SEQ ID NO:29, and a probe comprising the sequence of SEQ IDNO:30.

In another embodiment, the kit further comprises reagents for detectingchikungunya virus. In certain embodiments, the kit comprises at leastone set of primers comprising a forward primer and a reverse primercapable of amplifying at least a portion of a chikungunya virus genomecomprising an NSP2 target sequence, wherein the primers are not morethan about 40 nucleotides in length, wherein the set of primers isselected from the group consisting of: a) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:6 and a reverse primer comprisingthe sequence of SEQ ID NO:7; b) a forward primer comprising at least 10contiguous nucleotides of the nucleotide sequence of SEQ ID NO:6 and areverse primer comprising at least 10 contiguous nucleotides of thenucleotide sequence of SEQ ID NO:7; c) a forward primer comprising anucleotide sequence having at least 95% identity to the sequence of SEQID NO:6 and a reverse primer comprising a nucleotide sequence having atleast 95% identity to the sequence of SEQ ID NO:7, wherein the primer iscapable of hybridizing to and amplifying chikungunya virus nucleic acidsin the nucleic acid amplification assay; d) a forward primer and areverse primer comprising at least one nucleotide sequence that differsfrom the corresponding nucleotide sequence of the forward primer orreverse primer of the primer set of (a) in that the primer has up tothree nucleotide changes compared to the corresponding sequence, whereinthe primer is capable of hybridizing to and amplifying chikungunya virusnucleic acids in the nucleic acid amplification assay; and e) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(d).

Additionally, the kit may further comprises at least one probe fordetecting chikungunya virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:8; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:8, wherein theprobe is capable of hybridizing to and detecting the chikungunya virusRNA or an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:8 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the chikungunya virus RNA or an amplicon thereof.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, chemistry, biochemistry,recombinant DNA techniques and immunology, within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Fundamental Virology (D. M. Knipe, P. M. Howley, D. E. Griffin, R. A.Lamb, M. A. Martin, B. Roizman, S. E. Straus, eds.), Lippincott Williams& Wilkins; Fourth edition, 2001; Dengue Virus (Current Topics inMicrobiology and Immunology, A. L. Rothman, ed.), Springer, 1^(st)edition, 2009; Frontiers in Dengue Virus Research (K. A. Hanley and S.C. Weaver eds.), Caister Academic Press, 1^(st) edition, 2010; Handbookof Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwelleds., Blackwell Scientific Publications); T. E. Creighton, Proteins:Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al., Molecular Cloning: A Laboratory Manual (3^(rd)Edition, 2001); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

1. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a virus oligonucleotide” includes a mixture of two or moresuch oligonucleotides, and the like.

As used herein, the term “chikungunya virus” refers to members of theTogaviridae family of enveloped viruses with a single-strandedpositive-sense RNA genome (see, e.g., Singh et al. (2011) Rev. Med.Virol. 21(2):78-88; herein incorporated by reference in its entirety).The term chikungunya virus may include any strain of chikungunya virus,such as a West African strain, East/Central/South African (ECSA) strain,or Asian strain (including Indian or south east clades), which iscapable of causing disease in an animal or human subject. In particular,the term encompasses any subtype of chikungunya virus that causesdisease in humans, including strains R80422a and the S27 Petersfield. Alarge number of chikungunya isolates have been partially or completelysequenced. See, e.g., the Virus Pathogen Resource (website atviprbrc.org/brc/home.spg?decorator=toga) and the GenBank database, whichcontain complete sequences for chikungunya viruses.

As used herein, the term “Zika virus” refers to members of theFlaviviridae family of enveloped viruses with a single-strandedpositive-sense RNA genome (see, e.g., Marano et al. (2015) BloodTransfus. 5:1-6, Hayes (2009) Emerg. Infect. Dis. 15(9):1347-1350;herein incorporated by reference in their entireties). The term Zikavirus may include any strain of Zika virus which is capable of causingdisease in an animal or human subject, including, but not limited to,strains from Africa, Asia, Central and South America, and the Caribbeanand Pacific Islands. A large number of Zika virus isolates have beenpartially or completely sequenced. See, e.g., the Virus PathogenResource and the GenBank database, which contain complete sequences forZika viruses.

As used herein, the term “dengue virus” refers to members of theFlaviviridae family of enveloped viruses with a single-strandedpositive-sense RNA genome (see, e.g., Frontiers in Dengue VirusResearch, Hanley and Weaver (editors), Caister Academic Press, 2010).The term dengue virus may include any serotype of dengue virus, such asserotypes 1-5, which is capable of causing disease in an animal or humansubject. In particular, the term encompasses any subtype of dengue virusthat causes disease in humans, including strains DEN 1 Hawaii 1944, Den2 New Guinea C strain, DEN 3 strain H87, and DEN 4 strain H241. A largenumber of dengue isolates have been partially or completely sequenced.See, e.g., the Broad Institute Dengue Virus Portal (website atbroadinstitute.org/annotation/viral/Dengue/); the Dengue Virus Database(website at denguedb.org); the Virus Pathogen Resource (website atviprbrc.org/brc/home.do?decorator=flavi_dengue) and the GenBankdatabase, which contain complete sequences for dengue viruses, includingserotypes 1-4.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, oligonucleotide, protein, or polypeptide)such that the substance comprises the majority percent of the sample inwhich it resides. Typically in a sample, a substantially purifiedcomponent comprises 50%, preferably 80%-85%, more preferably 90-95% ofthe sample. Techniques for purifying polynucleotides oligonucleotidesand polypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide or oligonucleotide is anucleic acid molecule devoid, in whole or part, of sequences normallyassociated with it in nature; or a sequence, as it exists in nature, buthaving heterologous sequences in association therewith; or a moleculedisassociated from the chromosome.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide molecules. Two nucleic acid, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 50% sequence identity, preferably atleast about 75% sequence identity, more preferably at least about80%-85% sequence identity, more preferably at least about 90% sequenceidentity, and most preferably at least about 95%-98% sequence identityover a defined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used herein to include a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotidic backbones,for example, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms will be used interchangeably. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA:RNAhybrids, and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide.

A chikungunya virus polynucleotide, oligonucleotide, nucleic acid andnucleic acid molecule, as defined above, is a nucleic acid moleculederived from a dengue virus, including, without limitation, any of thevarious chikungunya virus strains. The molecule need not be physicallyderived from the particular isolate in question, but may besynthetically or recombinantly produced.

Nucleic acid and protein sequences for a number of chikungunya virusisolates are known. A representative chikungunya virus sequence ispresented in SEQ ID NO:26 of the Sequence Listing. Additionalrepresentative sequences, including RNA genomic sequences and sequencesof the non-structural polyprotein, the non-structural proteins (nsP1,nsP2, nsP3 and nsP4) produced by proteolytic processing of thenon-structural polyprotein, the structural polyprotein, and thestructural proteins (Capsid, E1, E2, E3, and 6K) produced by proteolyticprocessing of the structural polyprotein for virus isolates of variousgenotypes, including West African, East/Central/South African (ECSA),and Asian (Indian and south east clades) strains are listed in theNational Center for Biotechnology Information (NCBI) database. See, forexample, NCBI entries: Accession No. AF369024, NC_004162, AF490259,EF210157, JX088705, KR559498, KR559497, KT449801, KM923920, KJ689453,KJ689452, KJ451624, KJ451623, KJ796852, KJ796851, KJ796847, KJ796846,KJ796844, KC488650, HM067743, GU189061, HM045823, HM045822, HM045821,HM045820, HM045819, HM045818, HM045817, HM045816, HM045815, HM045814,HM045813, HM045812, HM045811, HM045810, HM045809, HM045808, HM045807,HM045806, HM045805, HM045804, HM045803, HM045802, HM045801, HM045800,HM045799, HM045798, HM045797, HM045796, HM045795, HM045794, HM045793,HM045792, HM045791, HM045790, HM045789, HM045788, HM045787, HM045786,HM045785, HM045784, HQ456255, HQ456254, HQ456253, HQ456252, HQ456251,GQ905863, AY726732, FJ807899, FJ807898, FJ807897, FJ807896, GQ428215,EF452494, EF452493, FJ959103, EU244823, EF012359, DQ443544, KF151175,KF151174, KP003813, KP003812, KP003811, KP003810, KP003809, KP003808,KP003807, KF872195, HE806461, HM067744, HQ848081, HQ848080, JQ067624,GU199353, GU199352, GU199351, GU199350, FJ807895, FJ807894, FJ807893,FJ807892, FJ807891, FJ807890, FJ807887, EU703762, EU703761, EU703760,EU703759, L37661, AB455494, AB455493, EU564335, EU564334, EF027141,EF027140, EF027139, EF027138, EF027137, EF027136, EF027135, EF027134,AY424803, AF339485, EU192143, EU192142, NC_004162, EU037962, KJ941050,KF151178, KF151177, KF151176, and AB678695; all of which sequences (asentered by the date of filing of this application) are hereinincorporated by reference. See also Lo Presti et al. (2014) Asian Pac.J. Trop. Med. 7(12):925-932, Weaver et al. (2015) Antiviral Res.120:32-39 and Caglioti et al. (2013) New Microbiol. 36(3):211-227 forsequence comparisons and a discussion of genetic diversity andphylogenetic analysis of chikungunya viruses.

A Zika virus polynucleotide, oligonucleotide, nucleic acid and nucleicacid molecule, as defined above, is a nucleic acid molecule derived froma Zika virus, including, without limitation, any of the various Zikavirus strains. The molecule need not be physically derived from theparticular isolate in question, but may be synthetically orrecombinantly produced.

Nucleic acid and protein sequences for a number of Zika virus isolatesare known. Representative Zika virus sequences are presented in SEQ IDNO:31 and SEQ ID NO:32 of the Sequence Listing. Additionalrepresentative sequences, including RNA genomic sequences and sequencesof the polyprotein and the non-structural proteins (NS1, NS2A, NS2B,NS3, NS4A, NS4B, and NS5) and the structural proteins (capsid protein(C), precursor membrane protein (prM), and envelope protein (E))produced by proteolytic processing of the polyprotein for virus isolatesof various strains are listed in the National Center for BiotechnologyInformation (NCBI) database. See, for example, NCBI entries: AccessionNo. NC_012532, KU647676, KU556802, KU509998, KU646828, KU646827,KU501217, KU501216, KU501215, KU365780, KU365779, KU365778, KU365777,KU312315, KU312314, KU312313, KU312312, KM078979, KM078978, KM078977,KM078976, KM078975, KM078974, KM078973, KM078972, KM078971, KM078970,KM078969, KM078968, KM078967, KM078966, KM078965, KM078964, KM078963,KM078962, KM078961, KM078960, KM078959, KM078958, KM078957, KM078956,KM078955, KM078954, KM078953, KM078952, KM078951, KM078950, KM078949,KM078948, KM078947, KM078946, KM078945, KM078944, KM078943, KM078942,KM078941, KM078940, KM078939, KM078938, KM078937, KM078936, KM078935,KM078934, KM078933, KM078932, KM078931, KM078930, KM078929, KR816336,KR816335, KR816334, KR816333, KM851039, KR815990, KR815989, KM851038,KM014700, KJ873161, KJ873160, KF993678, LC002520, KJ461621, KJ776791,KJ634273, KJ579442, KJ579441, KJ680135, KJ680134, KF383121, KF383120,KF383119, KF383118, KF383117, KF383116, KF383115, KF383114, KF383113,KF383112, KF383111, KF383110, KF383109, KF383108, KF383107, KF383106,KF383105, KF383104, KF383103, KF383102, KF383101, KF383100, KF383099,KF383098, KF383097, KF383096, KF383095, KF383094, KF383093, KF383092,KF383091, KF383090, KF383089, KF383088, KF383087, KF383086, KF383085,KF383084, KF383083, KF383082, KF383081, KF383080, KF383079, KF383078,KF383077, KF383076, KF383075, KF383074, KF383073, KF383072, KF383071,KF383070, KF383069, KF383068, KF383067, KF383066, KF383065, KF383064,KF383063, KF383062, KF383061, KF383060, KF383059, KF383058, KF383057,KF383056, KF383055, KF383054, KF383053, KF383053, KF383051, KF383050,KF383049, KF383048, KF383047, KF383046, KF383045, KF383044, KF383043,KF383042, KF383041, KF383040, KF383039, KF383038, KF383037, KF383036,KF383035, KF383034, KF383033, KF383032, KF383031, KF383030, KF383029,KF383028, KF383027, KF383026, KF383025, KF383024, KF383023, KF383022,KF383021, KF383020, KF383019, KF383018, KF383017, KF383016, KF383015,KF270887, KF270886, KF258813, JN860885, HQ234501, HQ234500, HQ234499,HQ234498, EU545988, KU681082, KU681081, KT200609, KU321639, KF268950,KF268949, KF268948, KP099610, KP099609, KM212967, KM212966, KM212965,KM212964, KM212963, KM212961, AB908162, AF013415, EU303241, AY632535,AF372422, EU074027, DQ859059, AY326412, and YP_002790881; all of whichsequences (as entered by the date of filing of this application) areherein incorporated by reference. See also Berthet et al. (2014) VectorBorne Zoonotic Dis. 14(12):862-865, Faye et al. (2014) PLoS Negl. Trop.Dis. 8(1):e2636, Zanluca et al. (2015) Mem. Inst. Oswaldo Cruz.110(4):569-572, Grard et al. (2014) PLoS Negl. Trop. Dis. 8(2):e2681,and Haddow et al. (2012) PLoS Negl. Trop. Dis. 6(2):e1477 for sequencecomparisons and a discussion of genetic diversity and phylogeneticanalysis of Zika viruses.

A dengue virus polynucleotide, oligonucleotide, nucleic acid and nucleicacid molecule, as defined above, is a nucleic acid molecule derived froma dengue virus, including, without limitation, any of the various denguevirus serotypes 1-4. The molecule need not be physically derived fromthe particular isolate in question, but may be synthetically orrecombinantly produced.

Nucleic acid sequences for a number of dengue virus isolates are known.Representative dengue virus sequences, including sequences of the5′-untranslated region (UTR) and coding region for the capsid protein Cfrom dengue virus isolates are listed in the National Center forBiotechnology Information (NCBI) database. See, for example, NCBIentries: Accession No. NC_001477, Accession No. NC_001474, Accession No.NC_001475, Accession No. NC_002640, Accession No. AB609588, AccessionNo. EU848545, Accession No. AB609589, Accession No. AF038403, AccessionNo. AF038402, Accession No. M29095, Accession No. M93130, Accession No.AB609590, Accession No. AB609591, Accession No. S66064, Accession No.AY947539, Accession No. JN559741, Accession No. JN559740, Accession No.JF357906, Accession No. HQ634199, Accession No. HQ541794, Accession No.EU076567, Accession No. EU076565, Accession No. EU076563, Accession No.EU076561, Accession No. JQ950328, Accession No. JN796245, Accession No.JN819424, Accession No. JN819422, Accession No. JN819414, Accession No.JN819412, Accession No. JN819406, Accession No. JN819417, Accession No.JN819415, Accession No. JN819409, Accession No. JN093514, Accession No.JF730055, Accession No. JN000937, Accession No. JF937647, Accession No.JN819406, Accession No. GQ868543; all of which sequences (as entered bythe date of filing of this application) are herein incorporated byreference. See also Weaver et al. (2009) Infect. Genet. Evol.9(4):523-540 and Rico-Hesse (2003) Adv. Virus Res. 59:315-341 forsequence comparisons and a discussion of genetic diversity andphylogenetic analysis of dengue viruses.

A polynucleotide “derived from” a designated sequence refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding, i.e.,identical or complementary to, a region of the designated nucleotidesequence. The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

As used herein, a “solid support” refers to a solid surface such as amagnetic bead, latex bead, microtiter plate well, glass plate, nylon,agarose, acrylamide, and the like.

As used herein, the term “target nucleic acid region” or “target nucleicacid” denotes a nucleic acid molecule with a “target sequence” to beamplified. The target nucleic acid may be either single-stranded ordouble-stranded and may include other sequences besides the targetsequence, which may not be amplified. The term “target sequence” refersto the particular nucleotide sequence of the target nucleic acid whichis to be amplified. The target sequence may include a probe-hybridizingregion contained within the target molecule with which a probe will forma stable hybrid under desired conditions. The “target sequence” may alsoinclude the complexing sequences to which the oligonucleotide primerscomplex and extended using the target sequence as a template. Where thetarget nucleic acid is originally single-stranded, the term “targetsequence” also refers to the sequence complementary to the “targetsequence” as present in the target nucleic acid. If the “target nucleicacid” is originally double-stranded, the term “target sequence” refersto both the plus (+) and minus (−) strands (or sense and anti-sensestrands).

The term “primer” or “oligonucleotide primer” as used herein, refers toan oligonucleotide that hybridizes to the template strand of a nucleicacid and initiates synthesis of a nucleic acid strand complementary tothe template strand when placed under conditions in which synthesis of aprimer extension product is induced, i.e., in the presence ofnucleotides and a polymerization-inducing agent such as a DNA or RNApolymerase and at suitable temperature, pH, metal concentration, andsalt concentration. The primer is preferably single-stranded for maximumefficiency in amplification, but may alternatively be double-stranded.If double-stranded, the primer can first be treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically effected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a template, and complexes by hydrogenbonding or hybridization with the template to give a primer/templatecomplex for initiation of synthesis by a polymerase, which is extendedby the addition of covalently bonded bases linked at its 3′ endcomplementary to the template in the process of DNA or RNA synthesis.Typically, viral nucleic acids are amplified using at least one set ofoligonucleotide primers comprising at least one forward primer and atleast one reverse primer capable of hybridizing to regions of a viralnucleic acid flanking the portion of the viral nucleic acid to beamplified. A forward primer for amplifying chikungunya virus iscomplementary to the 3′ end of the anti-genomic chikungunya virustemplate produced during replication or amplification of chikungunyavirus nucleic acids. A reverse primer for amplifying chikungunya virusis complementary to the 3′ end of the chikungunya virus positive sensegenomic RNA strand. A forward primer for amplifying Zika virus iscomplementary to the 3′ end of the anti-genomic Zika virus templateproduced during replication or amplification of Zika virus nucleicacids. A reverse primer for amplifying Zika virus is complementary tothe 3′ end of the Zika virus positive sense genomic RNA strand.

The term “amplicon” refers to the amplified nucleic acid product of aPCR reaction or other nucleic acid amplification process (e.g., ligasechain reaction (LGR), nucleic acid sequence based amplification (NASBA),transcription-mediated amplification (TMA), Q-beta amplification, stranddisplacement amplification, or target mediated amplification). Ampliconsmay comprise RNA or DNA depending on the technique used foramplification. For example, DNA amplicons may be generated by RT-PCR,whereas RNA amplicons may be generated by TMA/NASBA.

As used herein, the term “probe” or “oligonucleotide probe” refers to apolynucleotide, as defined above, that contains a nucleic acid sequencecomplementary to a nucleic acid sequence present in the target nucleicacid analyte. The polynucleotide regions of probes may be composed ofDNA, and/or RNA, and/or synthetic nucleotide analogs. Probes may belabeled in order to detect the target sequence. Such a label may bepresent at the 5′ end, at the 3′ end, at both the 5′ and 3′ ends, and/orinternally. The “oligonucleotide probe” may contain at least onefluorescer and at least one quencher. Quenching of fluorophorefluorescence may be eliminated by exonuclease cleavage of thefluorophore from the oligonucleotide (e.g., TaqMan assay) or byhybridization of the oligonucleotide probe to the nucleic acid targetsequence (e.g., molecular beacons). Additionally, the oligonucleotideprobe will typically be derived from a sequence that lies between thesense and the antisense primers when used in a nucleic acidamplification assay.

As used herein, the term “capture oligonucleotide” refers to anoligonucleotide that contains a nucleic acid sequence complementary to anucleic acid sequence present in the target nucleic acid analyte suchthat the capture oligonucleotide can “capture” the target nucleic acid.One or more capture oligonucleotides can be used in order to capture thetarget analyte. The polynucleotide regions of a capture oligonucleotidemay be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.By “capture” is meant that the analyte can be separated from othercomponents of the sample by virtue of the binding of the capturemolecule to the analyte. Typically, the capture molecule is associatedwith a solid support, either directly or indirectly.

It will be appreciated that the hybridizing sequences need not haveperfect complementarity to provide stable hybrids. In many situations,stable hybrids will form where fewer than about 10% of the bases aremismatches, ignoring loops of four or more nucleotides. Accordingly, asused herein the term “complementary” refers to an oligonucleotide thatforms a stable duplex with its “complement” under assay conditions,generally where there is about 90% or greater homology.

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer “hybridizes” with target (template), such complexes (or hybrids)are sufficiently stable to serve the priming function required by, e.g.,the DNA polymerase to initiate DNA synthesis.

As used herein, the term “binding pair” refers to first and secondmolecules that specifically bind to each other, such as complementarypolynucleotide pairs capable of forming nucleic acid duplexes. “Specificbinding” of the first member of the binding pair to the second member ofthe binding pair in a sample is evidenced by the binding of the firstmember to the second member, or vice versa, with greater affinity andspecificity than to other components in the sample. The binding betweenthe members of the binding pair is typically noncovalent. Unless thecontext clearly indicates otherwise, the terms “affinity molecule” and“target analyte” are used herein to refer to first and second members ofa binding pair, respectively.

The terms “specific-binding molecule” and “affinity molecule” are usedinterchangeably herein and refer to a molecule that will selectivelybind, through chemical or physical means to a detectable substancepresent in a sample. By “selectively bind” is meant that the moleculebinds preferentially to the target of interest or binds with greateraffinity to the target than to other molecules. For example, a DNAmolecule will bind to a substantially complementary sequence and not tounrelated sequences. An oligonucleotide that “specifically binds” to aparticular type of chikungunya virus, such as a particular genotype ofchikungunya virus (e.g., West African, East/Central/South African(ECSA), or Asian (Indian or south east clade) genotype), denotes anoligonucleotide, e.g., a primer, probe or a capture oligonucleotide,that binds to the particular chikungunya virus genotype, but does notbind to a sequence from other types of chikungunya viruses. Anoligonucleotide that “specifically binds” to a particular type of Zikavirus, such as a particular genotype of Zika virus, denotes anoligonucleotide, e.g., a primer, probe or a capture oligonucleotide,that binds to the particular Zika virus genotype, but does not bind to asequence from other types of Zika viruses.

The terms “selectively detects” or “selectively detecting” refer to thedetection of chikungunya virus, Zika virus, or dengue virus nucleicacids using oligonucleotides, e.g., primers, probes and/or captureoligonucleotides that are capable of detecting a particular viralnucleic acid, for example, by amplifying and/or binding to at least aportion of an RNA segment from a particular type of virus, such as aparticular virus genotype, but do not amplify and/or bind to sequencesfrom other types of viruses under appropriate hybridization conditions.

The “melting temperature” or “Tm” of double-stranded DNA is defined asthe temperature at which half of the helical structure of DNA is lostdue to heating or other dissociation of the hydrogen bonding betweenbase pairs, for example, by acid or alkali treatment, or the like. TheT_(m) of a DNA molecule depends on its length and on its basecomposition. DNA molecules rich in GC base pairs have a higher T_(m)than those having an abundance of AT base pairs. Separated complementarystrands of DNA spontaneously reassociate or anneal to form duplex DNAwhen the temperature is lowered below the T_(m). The highest rate ofnucleic acid hybridization occurs approximately 25 degrees C. below theT_(m). The T_(m) may be estimated using the following relationship:T_(m)=69.3+0.41(GC) % (Marmur et al. (1962) J. Mol. Biol. 5:109-118).

As used herein, a “biological sample” refers to a sample of cells,tissue, or fluid isolated from a subject, including but not limited to,for example, blood, plasma, serum, fecal matter, urine, bone marrow,bile, spinal fluid, lymph fluid, samples of the skin, externalsecretions of the skin, respiratory, intestinal, and genitourinarytracts, tears, saliva, milk, cells (e.g., epithelial and endothelialcells, fibroblasts, and macrophages), muscles, joints, organs (e.g.,liver, lung, spleen, thymus, kidney, brain, or lymph node), biopsies andalso samples of in vitro cell culture constituents including but notlimited to conditioned media resulting from the growth of cells andtissues in culture medium, e.g., recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,semiconductor nanoparticles, dyes, metal ions, metal sols, ligands(e.g., biotin, strepavidin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof which is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels which may be used in the practice of the invention include,but are not limited to, SYBR green, SYBR gold, a CAL Fluor dye such asCAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL FluorRed 610, and CAL Fluor Red 635, a Quasar dye such as Quasar 570, Quasar670, and Quasar 705, an Alexa Fluor such as Alexa Fluor 350, Alexa Fluor488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647,and Alexa Fluor 784, a cyanine dye such as Cy 3, Cy3.5, Cy5, Cy5.5, andCy7, fluorescein, 2′, 4′, 5′, 7′-tetrachloro-4-7-dichlorofluorescein(TET), carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX),tetramethyl rhodamine (TAMRA), FITC, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas red, luminol, NADPH, horseradishperoxidase (HRP), and α-β-galactosidase.

A “molecular beacon” probe is a single-stranded oligonucleotide,typically 25 to 40 bases-long, in which the bases on the 3′ and 5′ endsare complementary forming a “stem,” typically for 5 to 8 base pairs. Amolecular beacon probe forms a hairpin structure at temperatures at andbelow those used to anneal the primers to the template (typically belowabout 60° C.). The double-helical stem of the hairpin brings afluorophore (or other label) attached to the 5′ end of the probe inproximity to a quencher attached to the 3′ end of the probe. The probedoes not fluoresce (or otherwise provide a signal) in this conformation.If a probe is heated above the temperature needed to melt the doublestranded stem apart, or the probe hybridizes to a target nucleic acidthat is complementary to the sequence within the single-strand loop ofthe probe, the fluorophore and the quencher are separated, and thefluorophore fluoresces in the resulting conformation. Therefore, in aseries of PCR cycles the strength of the fluorescent signal increases inproportion to the amount of the molecular beacon that is hybridized tothe amplicon, when the signal is read at the annealing temperature.Molecular beacons of high specificity, having different loop sequencesand conjugated to different fluorophores, can be selected in order tomonitor increases in amplicons that differ by as little as one base(Tyagi, S. and Kramer, F. R. (1996), Nat. Biotech. 14:303 308; Tyagi, S.et al., (1998), Nat. Biotech. 16: 49 53; Kostrikis, L. G. et al.,(1998), Science 279: 1228 1229; all of which are herein incorporated byreference).

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; birds; and laboratory animals, including rodentssuch as mice, rats and guinea pigs, and the like. The term does notdenote a particular age. Thus, both adult and newborn individuals areintended to be covered.

2. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention is based on the discovery of reagents and methodsfor diagnosing infection caused by chikungunya and Zika viruses. Inparticular, the invention provides quantitative assays that can detectall lineages of chikungunya virus and Zika virus and distinguishchikungunya virus and Zika virus from each other as well as dengue virusand other arbovirus pathogens.

The methods are useful for detecting chikungunya and Zika viruses inbiological samples such as blood samples, including without limitation,in whole blood, serum and plasma. Thus, the methods can be used todiagnose chikungunya virus or Zika virus infection in a subject, as wellas to detect chikungunya virus or Zika virus contamination in donatedblood samples. Aliquots from individual donated samples or pooledsamples can be screened for the presence of chikungunya virus or Zikavirus and those samples or pooled samples contaminated with chikungunyavirus or Zika virus can be eliminated before they are combined. In thisway, a blood supply substantially free of chikungunya virus or Zikavirus contamination can be provided.

Chikungunya and Zika viruses can also be detected in other bodilyfluids, cells, or tissue samples in which the virus proliferates,including, but not limited to, saliva, cerebrospinal fluid (CSF), urine,amniotic fluid, dendritic cells, fibroblasts, keratinocytes, epithelialcells, endothelial cells, macrophages, and tissue samples obtained fromthe skin, liver, muscles, joints, spleen, lymph nodes, thymus, lung,brain, nerves, kidneys, or bone marrow. The, chikungunya and Zikaviruses can be specifically detected even in samples containing otherviruses and pathogens, such as dengue virus, West Nile virus, Japaneseencephalitis virus, tick-borne encephalitis virus, yellow fever virus,Saint Louis encephalitis virus, o'nyong-nyong virus, Semliki Forestvirus, mayaro virus, Ross River virus, Getah virus, Barmah Forest virus,Una virus, hepatitis C virus (HCV), Leptospira, Plasmodium species, RiftValley fever virus, and human immunodeficiency virus (HIV). Moreover,the methods described herein can be used to screen mosquitoes, primates,birds, cattle, rodents, and other hosts for chikungunya virus in orderto determine if a particular insect or animal population is infectedwith the chikungunya virus, thereby preventing further transmission andspread of viral infection. Furthermore, the methods described herein canbe used to screen mosquitoes, primates, rodents, and other hosts forZika virus in order to determine if a particular insect or animalpopulation is infected with the Zika virus, thereby preventing furthertransmission and spread of viral infection.

The methods use oligonucleotide reagents (e.g., oligonucleotide primersand probes) or a combination of reagents capable of detecting one ormore pathogenic chikungunya and/or Zika viruses in a single assay. Inone format, primer pairs and probes capable of detecting one or morepathogenic chikungunya or Zika viruses are used. For example, certainprimers and probes are from “conserved” regions of chikungunya virus andtherefore capable of detecting more than one pathogenic chikungunya,such as any combination of two or more chikungunya viruses that arepathogenic in humans, for example, two or more genotypes of chikungunya(e.g., both chikungunya West African and ECSA; West African and Asian;Asian and ECSA; or West African, Asian, and ECSA).

By way of example, the NSP2 gene of chikungunya virus includes conservedregions. Thus, primers and probes comprising sequences from theseconserved regions, or the corresponding regions in other pathogenicchikungunya viruses, may be useful in detecting multiple pathogenicchikungunya viruses.

Other primers and probes are highly selective for a particularchikungunya virus, selectively amplifying, detecting and/or binding to aparticular RNA segment from one of the virus genotypes. These highlyselective primers and probes can be used alone or in combination todetect one or more viruses in a single assay.

Thus, there are a number of assay designs that can be used to detecthuman pathogenic viral genotypes alone or in combination with eachother. In one embodiment, “conserved” primers (i.e., those primers thatamplify more than one chikungunya virus) can be used to detect one ormore of the chikungunya virus genotypes, as specified above. Forexample, conserved primers and probes can be used to amplify and detectmultiple viral genotypes. Alternatively, genotype-specific primers andprobe(s) can be used to achieve specificity. For example, a singlepathogenic genotype (e.g., West African, Asian, or ECSA strain) can beamplified with genotype-specific primers and detected with thecorresponding genotype-specific probes. One or more genotypes can beamplified and detected simultaneously by using a combination ofgenotype-specific primers and probes in a multiplex-type assay format.

Thus, the probes and primers may be designed from conserved nucleotideregions of the polynucleotides of interest or from non-conservednucleotide regions of the polynucleotide of interest. Generally, nucleicacid probes are developed from non-conserved or unique regions whenmaximum specificity is desired, and nucleic acid probes are developedfrom conserved regions when assaying for nucleotide regions that areclosely related to, for example, different chikungunya virus isolates.

Oligonucleotides for use in the assays described herein can be derived,for example, from the NSP2 gene sequences of chikungunya viruses.Representative sequences from chikungunya isolates are listed herein.Thus, primers and probes for use in detection of chikungunya virusinclude those derived from any one of the chikungunya West African,East/Central/South African (ECSA), and Asian (Indian and south eastclades) virus genotypes, including any pathogenic chikungunya virusstrain or isolate.

Representative sequences for a number of chikungunya virus isolates areknown. A representative chikungunya virus sequence is presented in SEQID NO:26 of the Sequence Listing. Additional representative sequences,including RNA genomic sequences and sequences of the non-structuralpolyprotein, the non-structural proteins (nsP1, nsP2, nsP3 and nsP4)produced by proteolytic processing of the non-structural polyprotein,the structural polyprotein, and the structural proteins (Capsid, E1, E2,E3, and 6K) produced by proteolytic processing of the structuralpolyprotein for virus isolates of various genotypes, including WestAfrican, East/Central/South African (ECSA), and Asian (Indian and southeast clades) strains are listed in the National Center for BiotechnologyInformation (NCBI) database. See, for example, NCBI entries: AccessionNo. AF369024, NC_004162, AF490259, EF210157, JX088705, KR559498,KR559497, KT449801, KM923920, KJ689453, KJ689452, KJ451624, KJ451623,KJ796852, KJ796851, KJ796847, KJ796846, KJ796844, KC488650, HM067743,GU189061, HM045823, HM045822, HM045821, HM045820, HM045819, HM045818,HM045817, HM045816, HM045815, HM045814, HM045813, HM045812, HM045811,HM045810, HM045809, HM045808, HM045807, HM045806, HM045805, HM045804,HM045803, HM045802, HM045801, HM045800, HM045799, HM045798, HM045797,HM045796, HM045795, HM045794, HM045793, HM045792, HM045791, HM045790,HM045789, HM045788, HM045787, HM045786, HM045785, HM045784, HQ456255,HQ456254, HQ456253, HQ456252, HQ456251, GQ905863, AY726732, FJ807899,FJ807898, FJ807897, FJ807896, GQ428215, EF452494, EF452493, FJ959103,EU244823, EF012359, DQ443544, KF151175, KF151174, KP003813, KP003812,KP003811, KP003810, KP003809, KP003808, KP003807, KF872195, HE806461,HM067744, HQ848081, HQ848080, JQ067624, GU199353, GU199352, GU199351,GU199350, FJ807895, FJ807894, FJ807893, FJ807892, FJ807891, FJ807890,FJ807887, EU703762, EU703761, EU703760, EU703759, L37661, AB455494,AB455493, EU564335, EU564334, EF027141, EF027140, EF027139, EF027138,EF027137, EF027136, EF027135, EF027134, AY424803, AF339485, EU192143,EU192142, NC_004162, EU037962, KJ941050, KF151178, KF151177, KF151176,and AB678695; all of which sequences (as entered by the date of filingof this application) are herein incorporated by reference. See also LoPresti et al. (2014) Asian Pac. J. Trop. Med. 7(12):925-932, Weaver etal. (2015) Antiviral Res. 120:32-39 and Caglioti et al. (2013) NewMicrobiol. 36(3):211-227 for sequence comparisons and a discussion ofgenetic diversity and phylogenetic analysis of chikungunya viruses.

The methods also use oligonucleotide reagents (e.g., oligonucleotideprimers and probes) or a combination of reagents capable of detectingone or more pathogenic Zika viruses in a single assay. In one format,primer pairs and probes capable of detecting one or more pathogenic Zikaviruses are used. For example, certain primers and probes are from“conserved” regions of Zika virus and therefore capable of detectingmore than one pathogenic Zika virus, such as any combination of two ormore Zika viruses that are pathogenic in humans, for example, two ormore genotypes of Zika virus (e.g., both African and Asian, African andCaribbean, or African, Asian and Central/South American).

By way of example, the region of the Zika virus genome from nucleotideposition 7332 to 7432, numbered relative to the reference sequence ofSEQ ID NO:32 of Zika virus strain MR766-NIID (GenBank: LC002520.1),includes conserved regions. Thus, primers and probes comprisingsequences from these conserved regions, or the corresponding regions inother pathogenic Zika viruses, may be useful in detecting multiplepathogenic Zika viruses.

Other primers and probes are highly selective for a particular Zikavirus, selectively amplifying, detecting and/or binding to a particularRNA segment from one of the virus genotypes. These highly selectiveprimers and probes can be used alone or in combination to detect one ormore viruses in a single assay.

Thus, there are a number of assay designs that can be used to detecthuman pathogenic viral genotypes alone or in combination with eachother. In one embodiment, “conserved” primers (i.e., those primers thatamplify more than one Zika virus) can be used to detect one or more ofthe Zika virus genotypes, as specified above. For example, conservedprimers and probes can be used to amplify and detect multiple viralgenotypes. Alternatively, genotype-specific primers and probe(s) can beused to achieve specificity. For example, a single pathogenic genotype(e.g., African, Asian, Caribbean, Pacific island, or Central/SouthAmerican strain) can be amplified with genotype-specific primers anddetected with the corresponding genotype-specific probes. One or moregenotypes can be amplified and detected simultaneously by using acombination of genotype-specific primers and probes in a multiplex-typeassay format.

Thus, the probes and primers may be designed from conserved nucleotideregions of the polynucleotides of interest or from non-conservednucleotide regions of the polynucleotide of interest. Generally, nucleicacid probes are developed from non-conserved or unique regions whenmaximum specificity is desired, and nucleic acid probes are developedfrom conserved regions when assaying for nucleotide regions that areclosely related in different Zika virus isolates.

Oligonucleotides for use in the assays described herein can be derived,for example, from gene sequences of Zika viruses. Representativesequences from Zika virus isolates are listed herein. Thus, primers andprobes for use in detection of Zika virus include those derived from anyZika virus genotype, including any pathogenic Zika virus strain orisolate, including strains from Africa, Asia, Central and South America,the Caribbean, and Pacific islands.

Representative sequences for a number of Zika virus isolates are known.Representative Zika virus sequences are presented in SEQ ID NO:31 andSEQ ID NO:32 of the Sequence Listing. Additional representativesequences, including RNA genomic sequences and sequences of thepolyprotein and the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A,NS4B, and NS5) and the structural proteins (capsid protein (C),precursor membrane protein (prM), and envelope protein (E)) produced byproteolytic processing of the polyprotein for virus isolates of variousstrains are listed in the National Center for Biotechnology Information(NCBI) database. See, for example, NCBI entries: Accession No.NC_012532, KU647676, KU556802, KU509998, KU646828, KU646827, KU501217,KU501216, KU501215, KU365780, KU365779, KU365778, KU365777, KU312315,KU312314, KU312313, KU312312, KM078979, KM078978, KM078977, KM078976,KM078975, KM078974, KM078973, KM078972, KM078971, KM078970, KM078969,KM078968, KM078967, KM078966, KM078965, KM078964, KM078963, KM078962,KM078961, KM078960, KM078959, KM078958, KM078957, KM078956, KM078955,KM078954, KM078953, KM078952, KM078951, KM078950, KM078949, KM078948,KM078947, KM078946, KM078945, KM078944, KM078943, KM078942, KM078941,KM078940, KM078939, KM078938, KM078937, KM078936, KM078935, KM078934,KM078933, KM078932, KM078931, KM078930, KM078929, KR816336, KR816335,KR816334, KR816333, KM851039, KR815990, KR815989, KM851038, KM014700,KJ873161, KJ873160, KF993678, LC002520, KJ461621, KJ776791, KJ634273,KJ579442, KJ579441, KJ680135, KJ680134, KF383121, KF383120, KF383119,KF383118, KF383117, KF383116, KF383115, KF383114, KF383113, KF383112,KF383111, KF383110, KF383109, KF383108, KF383107, KF383106, KF383105,KF383104, KF383103, KF383102, KF383101, KF383100, KF383099, KF383098,KF383097, KF383096, KF383095, KF383094, KF383093, KF383092, KF383091,KF383090, KF383089, KF383088, KF383087, KF383086, KF383085, KF383084,KF383083, KF383082, KF383081, KF383080, KF383079, KF383078, KF383077,KF383076, KF383075, KF383074, KF383073, KF383072, KF383071, KF383070,KF383069, KF383068, KF383067, KF383066, KF383065, KF383064, KF383063,KF383062, KF383061, KF383060, KF383059, KF383058, KF383057, KF383056,KF383055, KF383054, KF383053, KF383053, KF383051, KF383050, KF383049,KF383048, KF383047, KF383046, KF383045, KF383044, KF383043, KF383042,KF383041, KF383040, KF383039, KF383038, KF383037, KF383036, KF383035,KF383034, KF383033, KF383032, KF383031, KF383030, KF383029, KF383028,KF383027, KF383026, KF383025, KF383024, KF383023, KF383022, KF383021,KF383020, KF383019, KF383018, KF383017, KF383016, KF383015, KF270887,KF270886, KF258813, JN860885, HQ234501, HQ234500, HQ234499, HQ234498,EU545988, KU681082, KU681081, KT200609, KU321639, KF268950, KF268949,KF268948, KP099610, KP099609, KM212967, KM212966, KM212965, KM212964,KM212963, KM212961, AB908162, AF013415, EU303241, AY632535, AF372422,EU074027, DQ859059, AY326412, and YP_002790881; all of which sequences(as entered by the date of filing of this application) are hereinincorporated by reference. See also Berthet et al. (2014) Vector BorneZoonotic Dis. 14(12):862-865, Faye et al. (2014) PLoS Negl. Trop. Dis.8(1):e2636, Zanluca et al. (2015) Mem. Inst. Oswaldo Cruz.110(4):569-572, Grard et al. (2014) PLoS Negl. Trop. Dis. 8(2):e2681,and Haddow et al. (2012) PLoS Negl. Trop. Dis. 6(2):e1477 for sequencecomparisons and a discussion of genetic diversity and phylogeneticanalysis of Zika viruses.

Primers and probes for use in the assays herein are derived from thesesequences and are readily synthesized by standard techniques, e.g.,solid phase synthesis via phosphoramidite chemistry, as disclosed inU.S. Pat. Nos. 4,458,066 and 4,415,732, incorporated herein byreference; Beaucage et al., Tetrahedron (1992) 48:2223-2311; and AppliedBiosystems User Bulletin No. 13 (1 Apr. 1987). Other chemical synthesismethods include, for example, the phosphotriester method described byNarang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester methoddisclosed by Brown et al., Meth. Enzymol. (1979) 68:109. Poly(A) orpoly(C), or other non-complementary nucleotide extensions may beincorporated into oligonucleotides using these same methods.Hexaethylene oxide extensions may be coupled to the oligonucleotides bymethods known in the art. Cload et al., J. Am. Chem. Soc. (1991)113:6324-6326; U.S. Pat. No. 4,914,210 to Levenson et al.; Durand etal., Nucleic Acids Res. (1990) 18:6353-6359; and Horn et al., Tet. Lett.(1986) 27:4705-4708.

Additionally, nucleic acids can be obtained directly from thechikungunya or Zika virus in question. For example, the chikungunyavirus is available from the ATCC (ATCC Accession No. VR-64, chikungunyavirus from serum of patient from Tanganyika, East Africa, 1953). TheZika virus is available from the ATCC (ATCC Accession No. VR-84, Zikavirus strain MR 766 from the blood of an experimental forest sentinelrhesus monkey, Uganda, 1947).

Alternatively, chikungunya virus can be isolated from infected mosquitosor animals. Once obtained, the virus can be propagated using knowntechniques (see, e.g., Buckley et al. (1975) Acta Virol. 19(1):10-18).Generally, chikungunya viruses are grown in cell culture. For example,A. albopictus and A. aegypti mosquito cell lines can be used forisolation of chikungunya virus (see, e.g., Sudeep et al. (2009) In VitroCell Dev. Biol. Anim 45(9):491-495, Lee et al. (2015) PLoS Negl. Trop.Dis. 9(3):e0003544). Alternatively, several mammalian cell lines, suchas human embryonic lung (HEL) cells, African green monkey kidney (VERO)cells, or baby hamster kidney (BHK-21) cells, may also be used (see,e.g., Pyndiah et al. (2012) Med. Trop. 72 Spec No:63-65, Davis et al.(1971) Appl. Microbiol. 21(2):338-341). In addition, virus may beobtained from inoculated animals susceptible to infection (e.g.,monkeys, birds, cattle, and rodents).

Zika virus can be isolated from infected mosquitos (e.g., Aedesafricanus, Aedes apicoargenteus, Aedes luteocephalus, Aedes aegypti,Aedes vitattus, and Aedes furcifer). Once obtained, the virus can bepropagated using known techniques (see, e.g., Way et al. (1976) J. Gen.Virol. 30(1):123-130, Wong et al. (2013) PLoS Negl Trop Dis. 7(8):e2348,and Li et al. (2012) PLoS Negl Trop Dis. 6(8):e1792; herein incorporatedby reference in their entireties). For example, A. albopictus and A.aegypti mosquito cell lines can be used for isolation of Zika virus.Alternatively, mammalian cell lines, such as the rhesus monkey kidneyLLC-MK2 cell line can be used (Way et al., supra). In addition, virusmay be obtained from inoculated animals susceptible to infection (e.g.,primates and rodents).

An amplification method such as PCR or nucleic acid sequence basedamplification (NASBA) can be used to amplify polynucleotides from eitherchikungunya virus or Zika virus genomic RNA or cDNA derived therefrom.Alternatively, polynucleotides can be synthesized in the laboratory, forexample, using an automatic synthesizer.

Typically, the primer oligonucleotides are in the range of between10-100 nucleotides in length, such as 15-60, 20-40 and so on, moretypically in the range of between 20-40 nucleotides long, and any lengthbetween the stated ranges.

In certain embodiments, a chikungunya virus primer oligonucleotidecomprises a sequence selected from the group consisting of SEQ IDNOS:1-3, 6, and 7; or a fragment thereof comprising at least about 6contiguous nucleotides, preferably at least about 8 contiguousnucleotides, more preferably at least about 10-12 contiguousnucleotides, and even more preferably at least about 15-20 contiguousnucleotides; or a variant thereof comprising a sequence having at leastabout 80-100% sequence identity thereto, including any percent identitywithin this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. Changes tothe nucleotide sequences of SEQ ID NOS: 1-3, 6, and 7 may be introducedcorresponding to genetic variations in particular chikungunya virusstrains. In certain embodiments, up to three nucleotide changes,including 1 nucleotide change, 2 nucleotide changes, or three nucleotidechanges, may be made in a sequence selected from the group consisting ofSEQ ID NOS: 1-3, 6, and 7, wherein the oligonucleotide primer is capableof hybridizing to and amplifying a particular chikungunya virus targetnucleic acid.

In other embodiments, a Zika virus primer oligonucleotide comprises asequence selected from the group consisting of SEQ ID NOS:27-29; or afragment thereof comprising at least about 6 contiguous nucleotides,preferably at least about 8 contiguous nucleotides, more preferably atleast about 10-12 contiguous nucleotides, and even more preferably atleast about 15-22 contiguous nucleotides; or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto. Changes to the nucleotide sequences ofSEQ ID NOS:27-29 may be introduced corresponding to genetic variationsin particular Zika virus strains. In certain embodiments, up to threenucleotide changes, including 1 nucleotide change, 2 nucleotide changes,or three nucleotide changes, may be made in a sequence selected from thegroup consisting of SEQ ID NOS:27-29, wherein the oligonucleotide primeris capable of hybridizing to and amplifying a particular Zika virustarget nucleic acid.

The typical probe oligonucleotide is in the range of between 10-100nucleotides long, such as 10-60, 15-40, 18-30, and so on, and any lengthbetween the stated ranges. In certain embodiments, a chikungunya virusprobe oligonucleotide comprises a sequence selected from the groupconsisting of SEQ ID NOS:4, 5, and 8; or a fragment thereof comprisingat least about 6 contiguous nucleotides, preferably at least about 8contiguous nucleotides, more preferably at least about 10-12 contiguousnucleotides, and even more preferably at least about 15-20 contiguousnucleotides; or a variant thereof comprising a sequence having at leastabout 80-100% sequence identity thereto, including any percent identitywithin this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. Changes tothe nucleotide sequences of SEQ ID NOS: 4, 5, and 8 may be introducedcorresponding to genetic variations in particular chikungunya virusstrains. In certain embodiments, up to three nucleotide changes,including 1 nucleotide change, 2 nucleotide changes, or three nucleotidechanges, may be made in a sequence selected from the group consisting ofSEQ ID NOS: 4, 5, and 8, wherein the oligonucleotide probe is capable ofhybridizing to and detecting a particular chikungunya virus targetnucleic acid.

In other embodiments, a Zika virus probe oligonucleotide comprises asequence selected from the group consisting of SEQ ID NO:30; or afragment thereof comprising at least about 6 contiguous nucleotides,preferably at least about 8 contiguous nucleotides, more preferably atleast about 10-12 contiguous nucleotides, and even more preferably atleast about 15-21 contiguous nucleotides; or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto. Changes to the nucleotide sequences ofSEQ ID NO:30 may be introduced corresponding to genetic variations inparticular Zika virus strains. In certain embodiments, up to threenucleotide changes, including 1 nucleotide change, 2 nucleotide changes,or three nucleotide changes, may be made to the sequence of SEQ IDNO:30, wherein the oligonucleotide probe is capable of hybridizing toand detecting a particular Zika virus target nucleic acid.

Moreover, the oligonucleotides, particularly the probe oligonucleotides,may be coupled to labels for detection. There are several means knownfor derivatizing oligonucleotides with reactive functionalities whichpermit the addition of a label. For example, several approaches areavailable for biotinylating probes so that radioactive, fluorescent,chemiluminescent, enzymatic, or electron dense labels can be attachedvia avidin. See, e.g., Broken et al., Nucl. Acids Res. (1978) 5:363-384which discloses the use of ferritin-avidin-biotin labels; and Chollet etal., Nucl. Acids Res. (1985) 13:1529-1541 which discloses biotinylationof the 5′ termini of oligonucleotides via an aminoalkylphosphoramidelinker arm. Several methods are also available for synthesizingamino-derivatized oligonucleotides which are readily labeled byfluorescent or other types of compounds derivatized by amino-reactivegroups, such as isothiocyanate, N-hydroxysuccinimide, or the like, see,e.g., Connolly, Nucl. Acids Res. (1987) 15:3131-3139, Gibson et al.Nucl. Acids Res. (1987) 15:6455-6467 and U.S. Pat. No. 4,605,735 toMiyoshi et al. Methods are also available for synthesizingsulfhydryl-derivatized oligonucleotides, which can be reacted withthiol-specific labels, see, e.g., U.S. Pat. No. 4,757,141 to Fung etal., Connolly et al., Nucl. Acids Res. (1985) 13:4485-4502 and Spoat etal. Nucl. Acids Res. (1987) 15:4837-4848. A comprehensive review ofmethodologies for labeling DNA fragments is provided in Matthews et al.,Anal. Biochem. (1988) 169:1-25.

For example, oligonucleotides may be fluorescently labeled by linking afluorescent molecule to the non-ligating terminus of the molecule.Guidance for selecting appropriate fluorescent labels can be found inSmith et al., Meth. Enzymol. (1987) 155:260-301; Karger et al., Nucl.Acids Res. (1991) 19:4955-4962; Guo et al. (2012) Anal. Bioanal. Chem.402(10):3115-3125; and Molecular Probes Handbook, A Guide to FluorescentProbes and Labeling Technologies, 11^(th) edition, Johnson and Spenceeds., 2010 (Molecular Probes/Life Technologies). Fluorescent labelsinclude fluorescein and derivatives thereof, such as disclosed in U.S.Pat. No. 4,318,846 and Lee et al., Cytometry (1989) 10:151-164. Dyes foruse in the present invention include 3-phenyl-7-isocyanatocoumarin,acridines, such as 9-isothiocyanatoacridine and acridine orange,pyrenes, benzoxadiazoles, and stilbenes, such as disclosed in U.S. Pat.No. 4,174,384. Additional dyes include SYBR green, SYBR gold, YakimaYellow, Texas Red,3-(ε-carboxypentyl)-3′-ethyl-5,5′-dimethyloxa-carbocyanine (CYA);6-carboxy fluorescein (FAM); CAL Fluor Orange 560, CAL Fluor Red 610,Quasar Blue 670; 5,6-carboxyrhodamine-110 (R110); 6-carboxyrhodamine-6G(R6G); N′,N′,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA);6-carboxy-X-rhodamine (ROX); 2′, 4′, 5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET); 2′, 7′-dimethoxy-4′, 5′-6carboxyrhodamine (JOE); 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein(HEX); Dragonfly orange; ATTO-Tec; Bodipy; ALEXA; VIC, Cy3, and Cy5.These dyes are commercially available from various suppliers such asLife Technologies (Carlsbad, Calif.), Biosearch Technologies (Novato,Calif.), and Integrated DNA Technologies (Coralville, Iowa). Fluorescentlabels include fluorescein and derivatives thereof, such as disclosed inU.S. Pat. No. 4,318,846 and Lee et al., Cytometry (1989) 10:151-164, and6-FAM, JOE, TAMRA, ROX, HEX-1, HEX-2, ZOE, TET-1 or NAN-2, and the like.

Oligonucleotides can also be labeled with a minor groove binding (MGB)molecule, such as disclosed in U.S. Pat. Nos. 6,884,584, 5,801,155;Afonina et al. (2002) Biotechniques 32:940-944, 946-949; Lopez-Andreo etal. (2005) Anal. Biochem. 339:73-82; and Belousov et al. (2004) HumGenomics 1:209-217. Oligonucleotides having a covalently attached MGBare more sequence specific for their complementary targets thanunmodified oligonucleotides. In addition, an MGB group increases hybridstability with complementary DNA target strands compared to unmodifiedoligonucleotides, allowing hybridization with shorter oligonucleotides.

Additionally, oligonucleotides can be labeled with an acridinium ester(AE) using the techniques described below. Current technologies allowthe AE label to be placed at any location within the probe. See, e.g.,Nelson et al., (1995) “Detection of Acridinium Esters byChemiluminescence” in Nonisotopic Probing, Blotting and Sequencing,Kricka L. J. (ed) Academic Press, San Diego, Calif.; Nelson et al.(1994) “Application of the Hybridization Protection Assay (HPA) to PCR”in The Polymerase Chain Reaction, Mullis et al. (eds.) Birkhauser,Boston, Mass.; Weeks et al., Clin. Chem. (1983) 29:1474-1479; Berry etal., Clin. Chem. (1988) 34:2087-2090. An AE molecule can be directlyattached to the probe using non-nucleotide-based linker arm chemistrythat allows placement of the label at any location within the probe.See, e.g., U.S. Pat. Nos. 5,585,481 and 5,185,439.

In certain embodiments, molecular beacon probes may be used fordetection of viral target nucleic acids. Molecular beacons are hairpinshaped oligonucleotides with an internally quenched fluorophore.Molecular beacons typically comprise four parts: a loop of about 18-30nucleotides, which is complementary to the target nucleic acid sequence;a stem formed by two oligonucleotide regions that are complementary toeach other, each about 5 to 7 nucleotide residues in length, on eitherside of the loop; a fluorophore covalently attached to the 5′ end of themolecular beacon, and a quencher covalently attached to the 3′ end ofthe molecular beacon. When the beacon is in its closed hairpinconformation, the quencher resides in proximity to the fluorophore,which results in quenching of the fluorescent emission from thefluorophore. In the presence of a target nucleic acid having a regionthat is complementary to the strand in the molecular beacon loop,hybridization occurs resulting in the formation of a duplex between thetarget nucleic acid and the molecular beacon. Hybridization disruptsintramolecular interactions in the stem of the molecular beacon andcauses the fluorophore and the quencher of the molecular beacon toseparate resulting in a fluorescent signal from the fluorophore thatindicates the presence of the target nucleic acid sequence. See, e.g.,Guo et al. (2012) Anal. Bioanal. Chem. 402(10):3115-3125; Wang et al.(2009) Angew. Chem. Int. Ed. Engl. 48(5):856-870; and Li et al. (2008)Biochem. Biophys. Res. Commun. 373(4):457-461; herein incorporated byreference in their entireties.

Representative chikungunya virus primers and probes derived fromconserved regions of the NSP2 gene for use in the various assays areshown in Example 1 in Table 1. These oligonucleotide primers and probesare designed to detect all genotypes of chikungunya virus. Furthermore,these chikungunya virus primers and probes can be used in combinationwith primers and probes designed for detecting other pathogens inmultiplex assays. For example, oligonucleotide primers and probes usefulfor detecting dengue virus, including serotypes 1-4, are shown inExample 1 in Tables 4 and 5 (see also Waggoner et al. (2013) J. Clin.Microbiol. 51:2172-2181 and International Application Publication No. WO2014/055746A1; herein incorporated by reference in their entireties).These chikungunya virus primers and probes can be combined with thedengue virus and/or Zika virus primers and probes described herein toallow chikungunya virus to be detected simultaneously with dengue virusand/or Zika virus in a single multiplex assay.

Representative Zika virus primers and probes derived from conservedregions of the Zika viral genome for use in the various assays are shownin Example 2 in Table 6. These oligonucleotide primers and probes aredesigned to detect all genotypes of Zika virus. Furthermore, these Zikavirus primers and probes can be used in combination with primers andprobes designed for detecting other pathogens in multiplex assays. Forexample, chikungunya virus and/or dengue virus primers and probes can becombined with the Zika virus primers and probes described herein toallow Zika virus to be detected simultaneously with chikungunya virusand/or dengue virus in a single multiplex assay.

It is to be understood that the primers and probes described herein aremerely representative, and other oligonucleotides derived from variouspathogenic chikungunya virus, Zika virus, or dengue virus strains orother arbovirus pathogens will find use in the assays described herein.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence.By selection of appropriate conditions, the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. An oligonucleotide that “selectively hybridizes” to aparticular virus sequence from a particular viral genotype underhybridization conditions described below, denotes an oligonucleotide,e.g., a primer or probe oligonucleotide, that binds to the viralsequence of that particular virus genotype, but does not bind to asequence from a virus of a different genotype.

In one embodiment of the present invention, a nucleic acid molecule iscapable of hybridizing selectively to a target sequence under moderatelystringent hybridization conditions. In the context of the presentinvention, moderately stringent hybridization conditions allow detectionof a target nucleic acid sequence of at least 14 nucleotides in lengthhaving at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe. In another embodiment, such selectivehybridization is performed under stringent hybridization conditions.Stringent hybridization conditions allow detection of target nucleicacid sequences of at least 14 nucleotides in length having a sequenceidentity of greater than 90% with the sequence of the selected nucleicacid probe. Hybridization conditions useful for probe/targethybridization where the probe and target have a specific degree ofsequence identity, can be determined as is known in the art (see, forexample, Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).Hybrid molecules can be formed, for example, on a solid support, insolution, and in tissue sections. The formation of hybrids can bemonitored by inclusion of a reporter molecule, typically, in the probe.Such reporter molecules or detectable labels include, but are notlimited to, radioactive elements, fluorescent markers, and molecules towhich an enzyme-conjugated ligand can bind.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is well known (see, forexample, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rdEdition, 2001).

As explained above, the primers and probes may be used in polymerasechain reaction (PCR)-based techniques, such as RT-PCR, to detectchikungunya virus infection in biological samples. PCR is a techniquefor amplifying a desired target nucleic acid sequence contained in anucleic acid molecule or mixture of molecules. In PCR, a pair of primersis employed in excess to hybridize to the complementary strands of thetarget nucleic acid. The primers are each extended by a polymerase usingthe target nucleic acid as a template. The extension products becometarget sequences themselves after dissociation from the original targetstrand. New primers are then hybridized and extended by a polymerase,and the cycle is repeated to geometrically increase the number of targetsequence molecules. The PCR method for amplifying target nucleic acidsequences in a sample is well known in the art and has been describedin, e.g., Innis et al. (eds.) PCR Protocols (Academic Press, N Y 1990);Taylor (1991) Polymerase chain reaction: basic principles andautomation, in PCR: A Practical Approach, McPherson et al. (eds.) IRLPress, Oxford; Saiki et al. (1986) Nature 324:163; as well as in U.S.Pat. Nos. 4,683,195, 4,683,202 and 4,889,818, all incorporated herein byreference in their entireties.

In particular, PCR uses relatively short oligonucleotide primers whichflank the target nucleotide sequence to be amplified, oriented such thattheir 3′ ends face each other, each primer extending toward the other.The polynucleotide sample is extracted and denatured, preferably byheat, and hybridized with first and second primers that are present inmolar excess. Polymerization is catalyzed in the presence of the fourdeoxyribonucleotide triphosphates (dNTPs—dATP, dGTP, dCTP and dTTP)using a primer- and template-dependent polynucleotide polymerizingagent, such as any enzyme capable of producing primer extensionproducts, for example, E. coli DNA polymerase I, Klenow fragment of DNApolymerase I, T4 DNA polymerase, thermostable DNA polymerases isolatedfrom Thermus aquaticus (Taq), available from a variety of sources (forexample, Perkin Elmer), Thermus thermophilus (United StatesBiochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcuslitoralis (“Vent” polymerase, New England Biolabs). This results in two“long products” which contain the respective primers at their 5′ endscovalently linked to the newly synthesized complements of the originalstrands. The reaction mixture is then returned to polymerizingconditions, e.g., by lowering the temperature, inactivating a denaturingagent, or adding more polymerase, and a second cycle is initiated. Thesecond cycle provides the two original strands, the two long productsfrom the first cycle, two new long products replicated from the originalstrands, and two “short products” replicated from the long products. Theshort products have the sequence of the target sequence with a primer ateach end. On each additional cycle, an additional two long products areproduced, and a number of short products equal to the number of long andshort products remaining at the end of the previous cycle. Thus, thenumber of short products containing the target sequence growsexponentially with each cycle. Preferably, PCR is carried out with acommercially available thermal cycler, e.g., Perkin Elmer.

RNAs may be amplified by reverse transcribing the RNA into cDNA, andthen performing PCR (RT-PCR), as described above. Alternatively, asingle enzyme may be used for both steps as described in U.S. Pat. No.5,322,770, incorporated herein by reference in its entirety. RNA mayalso be reverse transcribed into cDNA, followed by asymmetric gap ligasechain reaction (RT-AGLCR) as described by Marshall et al. (1994) PCRMeth. App. 4:80-84.

Nucleic acid sequence based amplification (NASBA) is an isothermalRNA-specific amplification method that does not require thermal cyclinginstrumentation. RNA is initially reverse transcribed such that thesingle-stranded RNA target is copied into a double-stranded DNA moleculethat serves as a template for RNA transcription. Detection of theamplified RNA is typically accomplished either byelectrochemiluminescence or in real-time, for example, withfluorescently labeled molecular beacon probes. See, e.g., Lau et al.(2006) Dev. Biol. (Basel) 126:7-15; and Deiman et al. (2002) Mol.Biotechnol. 20(2):163-179.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are employed in molar excess to the target. The first probehybridizes to a first segment of the target strand, and the second probehybridizes to a second segment of the target strand, the first andsecond segments being contiguous so that the primary probes abut oneanother in 5′ phosphate-3′ hydroxyl relationship, and so that a ligasecan covalently fuse or ligate the two probes into a fused product. Inaddition, a third (secondary) probe can hybridize to a portion of thefirst probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion. If the target isinitially double stranded, the secondary probes also will hybridize tothe target complement in the first instance. Once the ligated strand ofprimary probes is separated from the target strand, it will hybridizewith the third and fourth probes which can be ligated to form acomplementary, secondary ligated product. It is important to realizethat the ligated products are functionally equivalent to either thetarget or its complement. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. Thistechnique is described more completely in EPA 320,308 to K. Backmanpublished Jun. 16, 1989 and EPA 439,182 to K. Backman et al., publishedJul. 31, 1991, both of which are incorporated herein by reference.

Other known methods for amplification of nucleic acids include, but arenot limited to self-sustained sequence replication (3SR) described byGuatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878 and J.Compton, Nature (1991) 350:91-92 (1991); Q-beta amplification; stranddisplacement amplification (as described in Walker et al., Clin. Chem.(1996) 42:9-13 and EPA 684,315; target mediated amplification, asdescribed in International Publication No. WO 93/22461, and the TaqMan™assay.

The fluorogenic 5′ nuclease assay, known as the TaqMan™ assay(Perkin-Elmer), is a powerful and versatile PCR-based detection systemfor nucleic acid targets. Primers and probes derived from conservedand/or non-conserved regions of the dengue virus genome in question canbe used in TaqMan™ analyses to detect the presence of infection in abiological sample. Analysis is performed in conjunction with thermalcycling by monitoring the generation of fluorescence signals. The assaysystem dispenses with the need for gel electrophoretic analysis, and iscapable of generating quantitative data allowing the determination oftarget copy numbers. For example, standard curves can be produced usingserial dilutions of previously quantified dengue viral suspensions. Astandard graph can be produced with copy numbers of each of the panelmembers against which sample unknowns can be compared.

The fluorogenic 5′ nuclease assay is conveniently performed using, forexample, AmpliTaq Gold™ DNA polymerase, which has endogenous 5′ nucleaseactivity, to digest an internal oligonucleotide probe labeled with botha fluorescent reporter dye and a quencher (see, Holland et al., Proc.Natl. Acad. Sci. USA (1991) 88:7276-7280; and Lee et al., Nucl. AcidsRes. (1993) 21:3761-3766). Assay results are detected by measuringchanges in fluorescence that occur during the amplification cycle as thefluorescent probe is digested, uncoupling the dye and quencher labelsand causing an increase in the fluorescent signal that is proportionalto the amplification of target nucleic acid.

The amplification products can be detected in solution or using solidsupports. In this method, the TaqMan™ probe is designed to hybridize toa target sequence within the desired PCR product. The 5′ end of theTaqMan™ probe contains a fluorescent reporter dye. The 3′ end of theprobe is blocked to prevent probe extension and contains a dye that willquench the fluorescence of the 5′ fluorophore. During subsequentamplification, the 5′ fluorescent label is cleaved off if a polymerasewith 5′ exonuclease activity is present in the reaction. Excision of the5′ fluorophore results in an increase in fluorescence that can bedetected.

For a detailed description of the TaqMan™ assay, reagents and conditionsfor use therein, see, e.g., Holland et al., Proc. Natl. Acad. Sci,U.S.A. (1991) 88:7276-7280; U.S. Pat. Nos. 5,538,848, 5,723,591, and5,876,930, all incorporated herein by reference in their entireties.

A class of quenchers, known as “Black Hole Quenchers” such as BHQ1 andBHQ2, can be used in the nucleic acid assays described above. Thesequenchers reduce background and improve signal to noise in PCR assays.These quenchers are described in, e.g., Johansson et al., J. Chem. Soc.(2002) 124:6950-6956 and are commercially available from BiosearchTechnologies (Novato, Calif.).

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a higher melt temperature than theprimer sequences. Preferably, the probe sequences have an estimated melttemperature that is about 10° C. higher than the melt temperature forthe amplification primer sequences. Hence, the primer sequences aregenerally shorter than the probe sequences. Typically, the primersequences are in the range of between 10-75 nucleotides long, moretypically in the range of 20-45. The typical probe is in the range ofbetween 10-50 nucleotides long, more typically 15-40 nucleotides inlength. Representative primers and probes useful in nucleic acidamplification assays are described above.

The dengue virus sequences described herein may also be used as a basisfor transcription-mediated amplification (TMA) assays. TMA is anisothermal, autocatalytic nucleic acid target amplification system thatcan provide more than a billion RNA copies of a target sequence, andthus provides a method of identifying target nucleic acid sequencespresent in very small amounts in a biological sample. For a detaileddescription of TMA assay methods, see, e.g., Hill (2001) Expert Rev.Mol. Diagn. 1:445-55; WO 89/1050; WO 88/10315; EPO Publication No.408,295; EPO Application No. 8811394-8.9; WO91/02818; U.S. Pat. Nos.5,399,491, 6,686,156, and 5,556,771, all incorporated herein byreference in their entireties.

Suitable DNA polymerases include reverse transcriptases, such as avianmyeloblastosis virus (AMV) reverse transcriptase (available from, e.g.,Seikagaku America, Inc.) and Moloney murine leukemia virus (MMLV)reverse transcriptase (available from, e.g., Bethesda ResearchLaboratories).

Promoters or promoter sequences suitable for incorporation in theprimers are nucleic acid sequences (either naturally occurring, producedsynthetically or a product of a restriction digest) that arespecifically recognized by an RNA polymerase that recognizes and bindsto that sequence and initiates the process of transcription whereby RNAtranscripts are produced. The sequence may optionally include nucleotidebases extending beyond the actual recognition site for the RNApolymerase which may impart added stability or susceptibility todegradation processes or increased transcription efficiency. Examples ofuseful promoters include those which are recognized by certainbacteriophage polymerases such as those from bacteriophage T3, T7 orSP6, or a promoter from E. coli. These RNA polymerases are readilyavailable from commercial sources, such as New England Biolabs andEpicentre.

Some of the reverse transcriptases suitable for use in the methodsherein have an RNAse H activity, such as AMV reverse transcriptase. Itmay, however, be preferable to add exogenous RNAse H, such as E. coliRNAse H, even when AMV reverse transcriptase is used. RNAse H is readilyavailable from, e.g., Bethesda Research Laboratories.

The RNA transcripts produced by these methods may serve as templates toproduce additional copies of the target sequence through theabove-described mechanisms. The system is autocatalytic andamplification occurs autocatalytically without the need for repeatedlymodifying or changing reaction conditions such as temperature, pH, ionicstrength or the like.

Detection may be done using a wide variety of methods, including directsequencing, hybridization with sequence-specific oligomers, gelelectrophoresis and mass spectrometry. These methods can useheterogeneous or homogeneous formats, isotopic or nonisotopic labels, aswell as no labels at all.

One method of detection is the use of target sequence-specificoligonucleotide probes described above. The probes may be used inhybridization protection assays (HPA). In this embodiment, the probesare conveniently labeled with acridinium ester (AE), a highlychemiluminescent molecule. See, e.g., Nelson et al. (1995) “Detection ofAcridinium Esters by Chemiluminescence” in Nonisotopic Probing, Blottingand Sequencing, Kricka L. J. (ed) Academic Press, San Diego, Calif.;Nelson et al. (1994) “Application of the Hybridization Protection Assay(HPA) to PCR” in The Polymerase Chain Reaction, Mullis et al. (eds.)Birkhauser, Boston, Mass.; Weeks et al., Clin. Chem. (1983)29:1474-1479; Berry et al., Clin. Chem. (1988) 34:2087-2090. One AEmolecule is directly attached to the probe using a non-nucleotide-basedlinker arm chemistry that allows placement of the label at any locationwithin the probe. See, e.g., U.S. Pat. Nos. 5,585,481 and 5,185,439.Chemiluminescence is triggered by reaction with alkaline hydrogenperoxide which yields an excited N-methyl acridone that subsequentlycollapses to ground state with the emission of a photon.

When the AE molecule is covalently attached to a nucleic acid probe,hydrolysis is rapid under mildly alkaline conditions. When theAE-labeled probe is exactly complementary to the target nucleic acid,the rate of AE hydrolysis is greatly reduced. Thus, hybridized andunhybridized AE-labeled probe can be detected directly in solution,without the need for physical separation.

HPA generally consists of the following steps: (a) the AE-labeled probeis hybridized with the target nucleic acid in solution for about 15 toabout 30 minutes. A mild alkaline solution is then added and AE coupledto the unhybridized probe is hydrolyzed. This reaction takesapproximately 5 to 10 minutes. The remaining hybrid-associated AE isdetected as a measure of the amount of target present. This step takesapproximately 2 to 5 seconds. Preferably, the differential hydrolysisstep is conducted at the same temperature as the hybridization step,typically at 50 to 70° C. Alternatively, a second differentialhydrolysis step may be conducted at room temperature. This allowselevated pHs to be used, for example in the range of 10-11, which yieldslarger differences in the rate of hydrolysis between hybridized andunhybridized AE-labeled probe. HPA is described in detail in, e.g., U.S.Pat. Nos. 6,004,745; 5,948,899; and 5,283,174, the disclosures of whichare incorporated by reference herein in their entireties.

In one example of a typical TMA assay, an isolated nucleic acid sample,suspected of containing a chikungunya virus target sequence, is mixedwith a buffer concentrate containing the buffer, salts, magnesium,nucleotide triphosphates, primers, dithiothreitol, and spermidine. Thereaction is optionally incubated at about 100° C. for approximately twominutes to denature any secondary structure. After cooling to roomtemperature, reverse transcriptase, RNA polymerase, and RNAse H areadded and the mixture is incubated for two to four hours at 37° C. Thereaction can then be assayed by denaturing the product, adding a probesolution, incubating 20 minutes at 60° C., adding a solution toselectively hydrolyze the unhybridized probe, incubating the reactionsix minutes at 60° C., and measuring the remaining chemiluminescence ina luminometer.

The methods of detection of the invention utilize a biological samplesuspected of containing chikungunya or Zika virus nucleic acids. Abiological sample may be pre-treated in any number of ways prior toassay for chikungunya or Zika virus nucleic acids. For instance, incertain embodiments, the sample may be treated to disrupt (or lyse) anyviral particles (virions), for example by treating the samples with oneor more detergents and/or denaturing agents (e.g., guanidinium agents).Nucleic acids may also be extracted from samples, for example, afterdetergent treatment and/or denaturing as described above. Total nucleicacid extraction may be performed using known techniques, for example bynon-specific binding to a solid phase (e.g., silica). See, e.g., U.S.Pat. Nos. 5,234,809, 6,849,431; 6,838,243; 6,815,541; and 6,720,166.

In certain embodiments, the target nucleic acids are separated fromnon-homologous nucleic acids using capture oligonucleotides immobilizedon a solid support. Such capture oligonucleotides contain nucleic acidsequences that are complementary to a nucleic acid sequence present inthe target chikungunya or Zika virus nucleic acid analyte such that thecapture oligonucleotide can “capture” the target nucleic acid. Captureoligonucleotides can be used alone or in combination to capture denguevirus nucleic acids. For example, multiple capture oligonucleotides canbe used in combination, e.g., 2, 3, 4, 5, 6, etc. different captureoligonucleotides can be attached to a solid support to capture targetchikungunya or Zika virus nucleic acids. In certain embodiments, one ormore capture oligonucleotides can be used to bind chikungunya or Zikavirus target nucleic acids either prior to or after amplification byprimer oligonucleotides and/or detection by probe oligonucleotides.

In one embodiment of the present invention the biological samplepotentially carrying target nucleic acids is contacted with a solidsupport in association with capture oligonucleotides. The captureoligonucleotides, which may be used separately or in combination, may beassociated with the solid support, for example, by covalent binding ofthe capture moiety to the solid support, by affinity association,hydrogen binding, or nonspecific association.

The capture oligonucleotides can include from about 5 to about 500nucleotides of a conserved region from a chikungunya or Zika virus,preferably about 10 to about 100 nucleotides, or more preferably about10 to about 60 nucleotides of the conserved region, or any integerwithin these ranges, such as a sequence including 18, 19, 20, 21, 22,23, 24, 25, 26 . . . 35 . . . 40, etc. nucleotides from the conservedregion of interest. In certain embodiments, the capture oligonucleotidecomprises a sequence selected from the group consisting of SEQ IDNOS:1-30, or a complement thereof. The capture oligonucleotide may alsobe phosphorylated at the 3′ end in order to prevent extension of thecapture oligonucleotide.

The capture oligonucleotide may be attached to the solid support in avariety of manners. For example, the oligonucleotide may be attached tothe solid support by attachment of the 3′ or 5′ terminal nucleotide ofthe probe to the solid support. More preferably, the captureoligonucleotide is attached to the solid support by a linker whichserves to distance the probe from the solid support. The linker isusually at least 10-50 atoms in length, more preferably at least 15-30atoms in length. The required length of the linker will depend on theparticular solid support used. For example, a six atom linker isgenerally sufficient when high cross-linked polystyrene is used as thesolid support.

A wide variety of linkers are known in the art which may be used toattach the oligonucleotide probe to the solid support. The linker may beformed of any compound which does not significantly interfere with thehybridization of the target sequence to the probe attached to the solidsupport. The linker may be formed of a homopolymeric oligonucleotidewhich can be readily added on to the linker by automated synthesis. Thehomopolymeric sequence can be either 5′ or 3′ to the virus-specificsequence. In one aspect of the invention, the capture oligonucleotidesinclude a homopolymer chain, such as, for example poly A, poly T, polyG, poly C, poly U, poly dA, poly dT, poly dG, poly dC, or poly dU inorder to facilitate attachment to a solid support. The homopolymer chaincan be from about 10 to about 40 nucleotides in length, or preferablyabout 12 to about 25 nucleotides in length, or any integer within theseranges, such as for example, 10 . . . 12 . . . 16, 17, 18, 19, 20, 21,22, 23, or 24 nucleotides. The homopolymer, if present, can be added tothe 3′ or 5′ terminus of the capture oligonucleotides by enzymatic orchemical methods. This addition can be made by stepwise addition ofnucleotides or by ligation of a preformed homopolymer. Captureoligonucleotides comprising such a homopolymer chain can be bound to asolid support comprising a complementary homopolymer. Alternatively,biotinylated capture oligonucleotides can be bound to avidin- orstreptavidin-coated beads. See, e.g., Chollet et al., supra.

Alternatively, polymers such as functionalized polyethylene glycol canbe used as the linker. Such polymers do not significantly interfere withthe hybridization of probe to the target oligonucleotide. Examples oflinkages include polyethylene glycol, carbamate and amide linkages. Thelinkages between the solid support, the linker and the probe arepreferably not cleaved during removal of base protecting groups underbasic conditions at high temperature.

The solid support may take many forms including, for example,nitrocellulose reduced to particulate form and retrievable upon passingthe sample medium containing the support through a sieve; nitrocelluloseor the materials impregnated with magnetic particles or the like,allowing the nitrocellulose to migrate within the sample medium upon theapplication of a magnetic field; beads or particles which may befiltered or exhibit electromagnetic properties; and polystyrene beadswhich partition to the surface of an aqueous medium. Examples of typesof solid supports for immobilization of the oligonucleotide probeinclude controlled pore glass, glass plates, polystyrene, avidin-coatedpolystyrene beads, cellulose, nylon, acrylamide gel and activateddextran.

In one embodiment, the solid support comprises magnetic beads. Themagnetic beads may contain primary amine functional groups, whichfacilitate covalent binding or association of the captureoligonucleotides to the magnetic support particles. Alternatively, themagnetic beads have immobilized thereon homopolymers, such as poly T orpoly A sequences. The homopolymers on the solid support will generallybe complementary to any homopolymer on the capture oligonucleotide toallow attachment of the capture oligonucleotide to the solid support byhybridization. The use of a solid support with magnetic beads allows fora one-pot method of isolation, amplification and detection as the solidsupport can be separated from the biological sample by magnetic means.

The magnetic beads or particles can be produced using standardtechniques or obtained from commercial sources. In general, theparticles or beads may be comprised of magnetic particles, although theycan also include other magnetic metal or metal oxides, whether inimpure, alloy, or composite form, as long as they have a reactivesurface and exhibit an ability to react to a magnetic field. Othermaterials that may be used individually or in combination with ironinclude, but are not limited to, cobalt, nickel, and silicon. A magneticbead suitable for use with the present invention includes magnetic beadscontaining poly dT groups marketed under the trade name Sera-Magmagnetic oligonucleotide beads by Seradyn, Indianapolis, Ind.

Next, the association of the capture oligonucleotides with the solidsupport is initiated by contacting the solid support with the mediumcontaining the capture oligonucleotides. In the preferred embodiment,the magnetic beads containing poly dT groups are hybridized with thecapture oligonucleotides that comprise poly dA contiguous with thecapture sequence (i.e., the sequence substantially complementary to achikungunya virus nucleic acid sequence) selected from the conservedsingle stranded region of the chikungunya virus genome. The poly dA onthe capture oligonucleotide and the poly dT on the solid supporthybridize thereby immobilizing or associating the captureoligonucleotides with the solid support.

In certain embodiments, the capture oligonucleotides are combined with abiological sample under conditions suitable for hybridization withtarget chikungunya virus nucleic acids prior to immobilization of thecapture oligonucleotides on a solid support. The captureoligonucleotide-target nucleic acid complexes formed are then bound tothe solid support. In other embodiments, a solid support with associatedcapture oligonucleotides is brought into contact with a biologicalsample under hybridizing conditions. The immobilized captureoligonucleotides hybridize to the target nucleic acids present in thebiological sample. Typically, hybridization of capture oligonucleotidesto the targets can be accomplished in approximately 15 minutes, but maytake as long as 3 to 48 hours.

The solid support is then separated from the biological sample, forexample, by filtering, centrifugation, passing through a column, or bymagnetic means. The solid support maybe washed to remove unboundcontaminants and transferred to a suitable container (e.g., a microtiterplate). As will be appreciated by one of skill in the art, the method ofseparation will depend on the type of solid support selected. Since thetargets are hybridized to the capture oligonucleotides immobilized onthe solid support, the target strands are thereby separated from theimpurities in the sample. In some cases, extraneous nucleic acids,proteins, carbohydrates, lipids, cellular debris, and other impuritiesmay still be bound to the support, although at much lower concentrationsthan initially found in the biological sample. Those skilled in the artwill recognize that some undesirable materials can be removed by washingthe support with a washing medium. The separation of the solid supportfrom the biological sample preferably removes at least about 70%, morepreferably about 90% and, most preferably, at least about 95% or more ofthe non-target nucleic acids present in the sample.

As is readily apparent, design of the assays described herein is subjectto a great deal of variation, and many formats are known in the art. Theabove descriptions are merely provided as guidance and one of skill inthe art can readily modify the described protocols, using techniqueswell known in the art.

The above-described assay reagents, including the primers and probes,and optionally capture oligonucleotides, a solid support with boundprobes, and/or reagents for performing nucleic acid amplification, suchas by RT-PCR or NASBA, can be provided in kits, with suitableinstructions and other necessary reagents, in order to conduct theassays for detecting chikungunya virus, Zika virus, or dengue virus, orany combination thereof, as described above. The kit will normallycontain in separate containers the primers and probes, controlformulations (positive and/or negative), and other reagents that theassay format requires. Instructions (e.g., written, CD-ROM, DVD, etc.)for carrying out the assay usually will be included in the kit. The kitcan also contain, depending on the particular assay used, other packagedreagents and materials (i.e., wash buffers, and the like). Standardassays, such as those described above, can be conducted using thesekits.

In certain embodiments, the kit comprises written instructions foridentifying the presence of chikungunya virus and at least one set ofprimers including a forward primer and a reverse primer capable ofamplifying at least a portion of a chikungunya virus genome, includingan NSP2 target sequence. The kit may further comprise writteninstructions for identifying the presence of the chikungunya,quantitating the virus, and/or serotyping chikungunya virus. The kit mayalso comprise reagents for performing reverse transcriptase polymerasechain reaction (RT-PCR), nucleic acid sequence based amplification(NASBA), transcription-mediated amplification (TMA), or a fluorogenic 5′nuclease assay. In one embodiment, the kit further comprisesoligonucleotide primers and probes for detecting dengue virus asdescribed herein.

In one embodiment, the kit comprises: written instructions foridentifying the presence of chikungunya virus; and at least one set ofprimers comprising a forward primer and a reverse primer capable ofamplifying at least a portion of a chikungunya virus genome comprisingan NSP2 target sequence, wherein the primers are not more than about 40nucleotides in length, wherein the set of primers is selected from thegroup consisting of: a) a forward primer comprising the nucleotidesequence of SEQ ID NO:6 and a reverse primer comprising the sequence ofSEQ ID NO:7; b) a forward primer comprising at least 10 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:6 and a reverseprimer comprising at least 10 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:7; c) a forward primer comprising a nucleotidesequence having at least 95% identity to the sequence of SEQ ID NO:6 anda reverse primer comprising a nucleotide sequence having at least 95%identity to the sequence of SEQ ID NO:7, wherein the primer is capableof hybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; d) a forward primer and a reverseprimer comprising at least one nucleotide sequence that differs from thecorresponding nucleotide sequence of the forward primer or reverseprimer of the primer set of (a) in that the primer has up to threenucleotide changes compared to the corresponding sequence, wherein theprimer is capable of hybridizing to and amplifying chikungunya virusnucleic acids in the nucleic acid amplification assay; and e) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(d).

Additionally, the kit may further comprises at least one probe fordetecting chikungunya virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:8; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:8, wherein theprobe is capable of hybridizing to and detecting the chikungunya virusRNA or an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:8 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the chikungunya virus RNA or an amplicon thereof.

In another embodiment, the kit further comprises reagents for detectingdengue virus, wherein the kit comprises a primer comprising the sequenceof SEQ ID NO:9, a primer comprising the sequence of SEQ ID NO:10, aprimer comprising the sequence of SEQ ID NO:11, a primer comprising thesequence of SEQ ID NO:12, a primer comprising the sequence of SEQ IDNO:13, a primer comprising the sequence of SEQ ID NO:14, a primercomprising the sequence of SEQ ID NO:15, and a primer comprising thesequence of SEQ ID NO:16.

Additionally, the kit may further comprises at least one probe fordetecting dengue virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:17, b) a probe comprising the sequence of SEQ IDNO:18, c) a probe comprising the sequence of SEQ ID NO:19, d) a probecomprising the sequence of SEQ ID NO:20, e) a probe comprising thesequence of SEQ ID NO:21, f) a probe comprising the sequence of SEQ IDNO:22, g) a probe comprising the sequence of SEQ ID NO:23, h) a probecomprising the sequence of SEQ ID NO:24, i) a probe comprising thesequence of SEQ ID NO:25, and j) a probe that differs from thecorresponding nucleotide sequence of a probe selected from the groupconsisting of (a)-(i) in that the probe has up to three nucleotidechanges compared to the corresponding sequence, wherein the probe iscapable of hybridizing to and detecting the dengue virus RNA or ampliconthereof. In one embodiment, a set of probes is used for detecting denguevirus in a biological sample, wherein the set of probes comprises aprobe comprising the sequence of SEQ ID NO:17, a probe comprising thesequence of SEQ ID NO:18, a probe comprising the sequence of SEQ IDNO:19, and a probe comprising the sequence of SEQ ID NO:20. In anotherembodiment, a set of probes is used for detecting dengue virus in abiological sample, wherein the set of probes comprises a probecomprising the sequence of SEQ ID NO:22, a probe comprising the sequenceof SEQ ID NO:23, a probe comprising the sequence of SEQ ID NO:24, and aprobe comprising the sequence of SEQ ID NO:25. In another embodiment, aset of primers and probes are used for detecting dengue virus in abiological sample comprising: a primer comprising the sequence of SEQ IDNO:9, a primer comprising the sequence of SEQ ID NO:10, a primercomprising the sequence of SEQ ID NO:11, a primer comprising thesequence of SEQ ID NO:12, a primer comprising the sequence of SEQ IDNO:13, a primer comprising the sequence of SEQ ID NO:14, a primercomprising the sequence of SEQ ID NO:15, a primer comprising thesequence of SEQ ID NO:16, a probe comprising the sequence of SEQ IDNO:17, a probe comprising the sequence of SEQ ID NO:18, a probecomprising the sequence of SEQ ID NO:19, and a probe comprising thesequence of SEQ ID NO:20.

In certain embodiments, the kit comprises written instructions foridentifying the presence of Zika virus and at least one set of primersincluding a forward primer and a reverse primer capable of amplifying atleast a portion of a Zika virus genome, including a conserved targetsequence (e.g., a sequence from the region of the Zika virus genomecorresponding to nucleotide position 7332 to 7432, numbered relative tothe reference sequence of SEQ ID NO:6, of Zika virus strain MR766-NIID).The kit may further comprise written instructions for identifying thepresence of the Zika virus, quantitating the virus, and/or serotypingthe Zika virus. The kit may also comprise reagents for performingreverse transcriptase polymerase chain reaction (RT-PCR), nucleic acidsequence based amplification (NASBA), transcription-mediatedamplification (TMA), or a fluorogenic 5′ nuclease assay. In oneembodiment, the kit further comprises probes for detecting Zika virus asdescribed herein.

In another embodiment, the kit comprises: written instructions foridentifying the presence of Zika virus; and at least one set of primerscomprising a forward primer and a reverse primer capable of amplifyingat least a portion of a Zika virus genome, wherein the primers are notmore than about 40 nucleotides in length, wherein the set of primers isselected from the group consisting of: a) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:27 and at least one reverse primercomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:28 and SEQ ID NO:29; b) a forward primer comprising at least10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:27 andat least one reverse primer comprising at least 10 contiguousnucleotides of a nucleotide sequence selected from the group consistingof SEQ ID NO:28 and SEQ ID NO:29; c) a forward primer comprising anucleotide sequence having at least 95% identity to the sequence of SEQID NO:27 and at least one reverse primer comprising a nucleotidesequence having at least 95% identity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:28 and SEQ ID NO:29, wherein theprimer is capable of hybridizing to and amplifying Zika virus nucleicacids in the nucleic acid amplification assay; d) a forward primer andat least one reverse primer comprising at least one nucleotide sequencethat differs from the corresponding nucleotide sequence of the forwardprimer or at least one reverse primer of the primer set of (a) in thatthe primer has up to three nucleotide changes compared to thecorresponding sequence, wherein the primer is capable of hybridizing toand amplifying Zika virus nucleic acids in the nucleic acidamplification assay; and e) a forward primer and at least one reverseprimer comprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(d). Inone embodiment, the kit comprises a forward primer comprising thenucleotide sequence of SEQ ID NO:27, a first reverse primer comprisingthe nucleotide sequence of SEQ ID NO:28, and a second reverse primercomprising the nucleotide sequence of SEQ ID NO:29.

Additionally, the kit may further comprises at least one probe fordetecting Zika virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:30; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:30, whereinthe probe is capable of hybridizing to and detecting the Zika virus RNAor an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:30 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the Zika virus RNA or an amplicon thereof. In one embodiment,the kit comprises a forward primer comprising the nucleotide sequence ofSEQ ID NO:27, a first reverse primer comprising the nucleotide sequenceof SEQ ID NO:28, a second reverse primer comprising the nucleotidesequence of SEQ ID NO:29, and a probe comprising the sequence of SEQ IDNO:30.

In certain embodiments, the kit further comprises reagents for detectingchikungunya virus in combination with Zika virus. In certainembodiments, the kit comprises at least one set of primers comprising aforward primer and a reverse primer capable of amplifying at least aportion of a chikungunya virus genome comprising an NSP2 targetsequence, wherein the primers are not more than about 40 nucleotides inlength, wherein the set of primers is selected from the group consistingof: a) a forward primer comprising the nucleotide sequence of SEQ IDNO:6 and a reverse primer comprising the sequence of SEQ ID NO:7; b) aforward primer comprising at least 10 contiguous nucleotides of thenucleotide sequence of SEQ ID NO:6 and a reverse primer comprising atleast 10 contiguous nucleotides of the nucleotide sequence of SEQ IDNO:7; c) a forward primer comprising a nucleotide sequence having atleast 95% identity to the sequence of SEQ ID NO:6 and a reverse primercomprising a nucleotide sequence having at least 95% identity to thesequence of SEQ ID NO:7, wherein the primer is capable of hybridizing toand amplifying chikungunya virus nucleic acids in the nucleic acidamplification assay; d) a forward primer and a reverse primer comprisingat least one nucleotide sequence that differs from the correspondingnucleotide sequence of the forward primer or reverse primer of theprimer set of (a) in that the primer has up to three nucleotide changescompared to the corresponding sequence, wherein the primer is capable ofhybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; and e) a forward primer and a reverseprimer comprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(d).Additionally, the kit may further comprises at least one probe fordetecting chikungunya virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:8; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:8, wherein theprobe is capable of hybridizing to and detecting the chikungunya virusRNA or an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:8 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the chikungunya virus RNA or an amplicon thereof.

In another embodiment, the kit further comprises reagents for detectingdengue virus in combination with Zika virus and/or chikungunya virus,wherein the kit comprises a primer comprising the sequence of SEQ IDNO:9, a primer comprising the sequence of SEQ ID NO:10, a primercomprising the sequence of SEQ ID NO:11, a primer comprising thesequence of SEQ ID NO:12, a primer comprising the sequence of SEQ IDNO:13, a primer comprising the sequence of SEQ ID NO:14, a primercomprising the sequence of SEQ ID NO:15, and a primer comprising thesequence of SEQ ID NO:16. Additionally, the kit may further comprises atleast one probe for detecting dengue virus in a biological sample,wherein the probe is selected from the group consisting of: a) a probecomprising the sequence of SEQ ID NO:17, b) a probe comprising thesequence of SEQ ID NO:18, c) a probe comprising the sequence of SEQ IDNO:19, d) a probe comprising the sequence of SEQ ID NO:20, e) a probecomprising the sequence of SEQ ID NO:21, f) a probe comprising thesequence of SEQ ID NO:22, g) a probe comprising the sequence of SEQ IDNO:23, h) a probe comprising the sequence of SEQ ID NO:24, i) a probecomprising the sequence of SEQ ID NO:25, and j) a probe that differsfrom the corresponding nucleotide sequence of a probe selected from thegroup consisting of (a)-(i) in that the probe has up to three nucleotidechanges compared to the corresponding sequence, wherein the probe iscapable of hybridizing to and detecting the dengue virus RNA or ampliconthereof. In one embodiment, a set of probes is used for detecting denguevirus in a biological sample, wherein the set of probes comprises aprobe comprising the sequence of SEQ ID NO:17, a probe comprising thesequence of SEQ ID NO:18, a probe comprising the sequence of SEQ IDNO:19, and a probe comprising the sequence of SEQ ID NO:20. In anotherembodiment, a set of probes is used for detecting dengue virus in abiological sample, wherein the set of probes comprises a probecomprising the sequence of SEQ ID NO:22, a probe comprising the sequenceof SEQ ID NO:23, a probe comprising the sequence of SEQ ID NO:24, and aprobe comprising the sequence of SEQ ID NO:25. In another embodiment, aset of primers and probes are used for detecting dengue virus in abiological sample comprising: a primer comprising the sequence of SEQ IDNO:9, a primer comprising the sequence of SEQ ID NO:10, a primercomprising the sequence of SEQ ID NO:11, a primer comprising thesequence of SEQ ID NO:12, a primer comprising the sequence of SEQ IDNO:13, a primer comprising the sequence of SEQ ID NO:14, a primercomprising the sequence of SEQ ID NO:15, a primer comprising thesequence of SEQ ID NO:16, a probe comprising the sequence of SEQ IDNO:17, a probe comprising the sequence of SEQ ID NO:18, a probecomprising the sequence of SEQ ID NO:19, and a probe comprising thesequence of SEQ ID NO:20.

3. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Clinical Evaluation of a Single-Reaction Real-Time RT-PCR forPan-Dengue and Chikungunya Virus Detection

Here, we describe the design and analytical and clinical evaluation of amultiplex assay for detecting Chikungunya and Dengue viruses, includinga clinical comparison with reference molecular testing performed inNicaragua.

Materials and Methods

Ethics Statement. Protocols for the collection and testing of samplesfrom Nicaraguan pediatric dengue and chikungunya cases were reviewed andapproved by the Institutional Review Boards (IRB) of the University ofCalifornia, Berkeley, the Nicaraguan Ministry of Health, and StanfordUniversity.

rRT-PCR Design and Performance. Design of the pan-DENV assay has beendescribed previously (Waggoner et al. (2013) J. Clin. Microbiol.51:2172-2181 and International Application Publication No. WO2014/055746A1; herein incorporated by reference in their entireties). Todesign the CHIKV rRT-PCR, all CHIKV complete genome sequences (n=130)available in GenBank (accessed in April, 2013) were aligned usingMegAlign software (DNAStar). A consensus sequence was generated thatidentified bases conserved across ≥95% of available sequences. Primersand probes were designed from the consensus sequence using Primer3software. CHIKV primers and probes were tested in silico using BLASTn tosearch the National Center for Biotechnology Information (NCBI)nucleotide database. Following an initial evaluation of three primersets, primers and probes targeting a region of the nsp2 gene (Table 1)were selected for improved analytical sensitivity (data not shown). Toconfirm that the selected primers and probe matched viral strainscirculating in the Western Hemisphere, a second CHIKV consensus sequencewas generated using all complete genome sequences deposited as of June,2015.

The pan-DENV-CHIKV rRT-PCR reactions were performed in a total volume of25 μL using the SuperScript III Platinum One-Step qRT-PCR kit (LifeTechnologies) and 5 μL of eluate. CHIKV primer and probe concentrationsin the final reaction mixture are listed in Table 1. DENV primers andprobes are shown in Tables 4 and 5. Concentrations of DENV primers andprobes in the final reaction mixture were the same as those previouslyreported (Waggoner, supra). Analytical evaluation was performed on aRotor-Gene Q instrument at Stanford University (RGQ, Qiagen), and theclinical evaluation was performed on a CFX96 instrument (Bio-Rad) at theNational Virology Laboratory in Managua, Nicaragua. Cycling conditionswere the following: 52° C. for 15 minutes; 94° C. for 2 minutes; 45cycles of 94° C. for 15 seconds, 55° C. for 40 seconds, and 68° C. for20 seconds. Each run was performed with a negative control (no template)and positive controls for DENV and CHIKV. Signal was acquired at 55° C.,and analysis was performed on the linear scale. Thresholds were setmanually on each instrument and used for analysis of each run (0.025 and0.1 on the RGQ, 500 and 100 on the CFX96 for DENV and CHIKV,respectively). For both targets, any exponential curve crossing thisthreshold was considered positive. All results after cycle 40 wereevaluated individually by two authors (JJW and GB).

Control Nucleic Acids and Reference Material. Quantitated,positive-sense, single-stranded DNA (ssDNA) oligonucleotides containingthe target sequence for each DENV serotype and CHIKV were synthesized(Eurofins MWG Operon) and used in the analytical characterization of thepan-DENV-CHIKV rRT-PCR. The specificity of the pan-DENV-CHIKV rRT-PCRwas evaluated by testing genomic RNA from the following viruses: DENV-1Hawaii 1944, DENV-2 New Guinea C strain, DENV-3 strain H87, and DENV-4strain H241, CHIKV (strain R80422a provided by the CDC Division ofVector Borne Diseases and the S27 Petersfield strain from VircellMicrobiologists, Granada, Spain), West Nile (4 strains), Japaneseencephalitis, tick-borne encephalitis, yellow fever (two strains), SaintLouis encephalitis, Zika, o'nyong-nyong (ONNV, strain MP30) (Seymour etal. (2013) Am. J. Trop. Med. Hyg. 88:1170-1179), Semliki Forest, mayaro,Ross River, Getah, Barmah Forest, and Una (Waggoner et al. (2013) J.Clin. Microbiol. 51:2172-2181; Waggoner et al. (2014) J. Clin.Microbiol. 52:2011-2018). An additional 50 domestic (USA) samples withdetectable hepatitis C virus (HCV) RNA were extracted and tested.

Analytical Characterization. The analytical evaluation of thepan-DENV-CHIKV rRT-PCR was performed according to publishedrecommendations (17) and as previously described (Waggoner et al. (2013)J. Clin. Microbiol. 51:2172-2181; Waggoner et al. (2014) J. Clin.Microbiol. 52:2011-2018). Briefly, for each DENV serotype and CHIKV,linearity studies were performed using serial 10-fold dilutions ofquantified ssDNA. Four replicates of each dilution from 7.0 log₁₀copies/μL to 1 copy/μL were tested on a single run. The linear range wasestablished by fitting a best-fit line to the data by regressionanalysis and included the range where the R² value was ≥0.99. Toestablish the lower limit of 95% detection (95% LLOD), ten replicates offive 2-fold dilutions from 50 to 3.12 copies/μL were tested on a singlerun. The 95% LLOD was then calculated using probit analysis.

Clinical Samples and Nucleic Acid Extraction. Pre-collected andde-identified serum samples from 182 Nicaraguan children with suspecteddengue or chikungunya were used for the clinical evaluation ofpan-DENV-CHIKV rRT-PCR. Samples were collected during 2013 and 2014 aspart of two ongoing studies: the Nicaraguan Pediatric Dengue CohortStudy and a hospital-based dengue study, both based in Managua. Studydesign and methods for both of these studies have been described(Hammond et al. (2005) Am. J. Trop. Med. Hyg. 73:1063; Harris et al.(2000) Am. J. Trop. Med. Hyg. 63:5-11; Kuan et al. (2009) Am. J.Epidemiol. 170:120-129). RNA extractions were performed using the QIAampViral RNA Mini Kit (Qiagen) with 140 μL of serum using and an elutionvolume of 60 μL.

Reference Testing

All serum samples were tested for DENV RNA using a hemi-nested RT-PCR(Lanciotti et al. (1992) J. Clin. Micobiol. 30:545-551). Acute andconvalescent serum samples were also tested for anti-DENV antibodies byIgM MAC-ELISA and Inhibition ELISA, which were interpreted as previouslydescribed (Gordon et al. (2013) PLoS Negl. Trop. Dis. 7:e2462).Eighty-one serum samples, collected in 2014, were tested for CHIKV usingan rRT-PCR, which maintained a protocol from a published assay(Lanciotti et al. (2007) Emerg. Infect. Dis. 13:764-767) but substitutedprimers and probes designed to detect Asian and East-Central-SouthAfrican genotypes of CHIKV. This will be referred to as the comparatorCHIKV rRT-PCR. Primer and probe sequences for the comparator CHIKVrRT-PCR are as follows:

forward primer: CHIKV 3855, (SEQ ID NO: 1) GAGCATACGGTTACGCAGATAG;reverse primers: CHIKV 3957c-a, (SEQ ID NO: 2) TACTGGTGATACATGGTGGTTTCand CHIKV 3957c-b, (SEQ ID NO: 3) TGCTGGTGACACATGGTGGTTTC; probes:CHIKV 3886 FAM-a, (SEQ ID NO: 4) FAM-ACGAGTAATCTGCGTACTGGGACGTA-BHQ1 andCHIKV 3886 FAM-b, (SEQ ID NO: 5) FAM-ACGAGTCATCTGCGTATTGGGACGCA-BHQ1.

Statistics. Kappa statistics were calculated with GraphPad software(GraphPad) and used to compare RT-PCR results. Probit analysis wasperformed using SPSS (IBM) to determine the 95% LLOD for thepan-DENV-CHIKV rRT-PCR.

Results

Analytical Evaluation. Using serial 10-fold dilutions of ssDNA, thelinear range of the pan-DENV-CHIKV rRT-PCR for each DENV serotype andCHIKV extended from 7.0 to 2.0 log₁₀ copies/μL. The 95% LLOD wascalculated to be 12.0 copies/μL for DENV-1, 7.9 for DENV-2, 12.9 forDENV-3, 37.4 for DENV-4, and 15.3 for CHIKV.

Specificity. Amplification of genomic RNA from ONNV was observed in theCHIKV channel of the pan-DENV-CHIKV rRT-PCR. This generated a signalwith reduced fluorescence compared to CHIKV genomic RNA controls (datanot shown). Amplification of the DENV and CHIKV controls was detected inthe appropriate channels without any cross-reaction. No amplificationwas observed when the pan-DENV-CHIKV rRT-PCR was performed using genomicRNA from the aforementioned viruses (see Material and Methods) orextracted nucleic acids from 50 patients with HCV (mean HCV viral load,6.10 log₁₀ IU/mL; standard deviation 0.76).

Clinical Samples. Sera from 182 Nicaraguan children with suspecteddengue or chikungunya were tested for DENV using the pan-DENV-CHIKVrRT-PCR and hemi-nested DENV RT-PCR. Results of this comparison areshown in Table 2. The two assays showed very good agreement (kappastatistic, 0.91). Seventy-five patients tested positive for DENV usingthe hemi-nested RT-PCR, including patients with DENV-1 (n=44), DENV-2(n=19), and DENV-3 (n=12). Seventy-four (98.7%) patients had detectableDENV RNA using the pan-DENV-CHIKV rRT-PCR. A single patient testedpositive for DENV-1 in the hemi-nested RT-PCR but was not detected inthe pan-DENV-CHIKV rRT-PCR. For an additional seven patients, DENV RNAcould only be detected using the pan-DENV-CHIKV rRT-PCR. Five of theseseven patients also tested positive for DENV by IgM MAC-ELISA (n=4) orInhibition ELISA (n=1).

Serum samples from 81 children, collected in 2014, were also tested forCHIKV using the comparator CHIKV rRT-PCR (Table 3). CHIKV test resultsdemonstrated complete agreement between the pan-DENV-CHIKV andcomparator CHIKV rRT-PCRs (kappa statistic, 1.0). CHIKV RNA was notdetected in any of the remaining 101 serum samples (collected in 2013)using the pan-DENV-CHIKV rRT-PCR and no co-infections were identified.

Discussion

In the current study, we describe the development and analytical andclinical evaluation of the pan-DENV-CHIKV rRT-PCR. This assaydemonstrated excellent agreement with separate molecular comparators forDENV and CHIKV. In addition, the pan-DENV-CHIKV rRT-PCR requires onlyone reaction per sample instead of the three reactions and gelelectrophoresis that are needed for the comparator assays. In thisstudy, the pan-DENV-CHIKV rRT-PCR did not significantly improve DENVdetection compared to the hemi-nested RT-PCR, which has been shown forthe pan-DENV assay previously (Waggoner et al. (2013) J. Clin.Microbiol. 51:2172-2181). However, the current study was not powered todemonstrate superiority. Of the 97 patients in this study who testednegative for DENV using the hemi-nested RT-PCR and had availableserological results, only 12 patients (12.4%) had results consistentwith a recent DENV infection. Five of these patients were detected usingthe pan-DENV CHIKV assay.

Multiplex RT-PCRs for the detection of DENV and CHIKV have been reportedin the literature previously (Cecilia et al. (2015) Arch. Virol.160:323-327; Dash et al. (2008) Diagn. Microbiol. Infect. Dis. 62:52-57;Mishra et al. (2011) Diagn. Microbiol. Infect. Dis. 71:118-125; Naze etal. (2009) J. Virol. Meth. 162:1; Pongsiri et al. (2012) Asian Pac. J.Trop. Med. 5:342-346; Saha et al. (2013) J. Virol. Methods 193:521-524).However, only three groups have described single-reaction real-timeassays for DENV and CHIKV (Cecilia et al., supra; Naze et al., supra;Pongsiri et al., supra), whereas others reported modified conventionalRT-PCRs that require gel electrophoresis for detection (Dash et al.,supra; Mishra et al., supra; Saha et al., supra). All multiplex rRT-PCRsappear to perform well, as reported, though most primer and probesequences contain mismatches when aligned to consensus DENV and CHIKVsequences that include contemporary viruses obtained globally (seeMaterials and Methods). Furthermore, in these studies less commoncomparator assays were used as reference or the exact molecularcomparator was unclear. These factors complicated the evaluation of thedifferent multiplex rRT-PCRs reported in the literature, and made themunsuitable for use as reference molecular assays for these viruses. Thegoal of our study was, therefore, to design an rRT-PCR for CHIKV thatwould be compatible with the pan-DENV assay, an assay that has excellentclinical and analytical performance characteristics and that has beenextensively evaluated in single- and multiplex formats (Waggoner et al.(2013) J. Clin. Microbiol. 51:2172-2181, Waggoner et al. (2014) J. Clin.Microbiol. 52:2011-2018, Waggoner et al. (2013) J. Clin. Microbiol.51:3418-3420). The resulting pan-DENV-CHIKV rRT-PCR was then compared towidely used, reference assays for DENV and CHIKV in Nicaragua, which isexperiencing the transmission of both viruses.

Although the pan-DENV-CHIKV rRT-PCR demonstrated very good analyticalperformance against all DENV serotypes and CHIKV, our study was limitedby the absence of DENV-4 in clinical samples. DENV-4 was not circulatingduring the 2013 and 2014 DENV seasons in Managua, and it has rarely beenidentified in clinical dengue cases in Managua over the past 10 years(OhAinle et al. (2011) Sci. Transl. Med. 3:114ra128). During theanalytical evaluation, the CHIKV rRT-PCR amplified genomic RNA from areference ONNV isolate. The in silico evaluation of our primers andprobe predicted two to four mismatches with ONNV, though only fourcomplete ONNV genomes were available for analysis. However, thesignificance of this cross-reaction remains unclear given the rarity ofONNV infections. Finally, the CHIKV rRT-PCR was designed from analignment performed prior to the emergence of CHIKV in the WesternHemisphere. The alignment was re-run using all complete genomesavailable as of June, 2015, and no new strains were identified withmutations in the primer and probe sequences.

In conclusion, we report the development and evaluation of thepan-DENV-CHIKV rRT-PCR. This assay demonstrated very good agreement withindividual molecular comparators for DENV and CHIKV. Thesingle-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR,combined with sensitive detection of both viruses, has the potential toimprove detection while decreasing test costs and streamlining molecularworkflow.

TABLE 1 CHIKV primer and probe sequences includedin the pan-DENV-CHIKV rRT-PCR. Concentra- Loca- Name Sequence (5′→3′)tion* tion** CHIKV CATCTGCACYCAAGT 200 nM 2578-2598 Forward GTACCA(SEQ ID NO: 6) CHIKV GCGCATTTTGCC 200 nM 2654-2674 Reverse TTCGTAATG(SEQ ID NO: 7) CHIKV GCGGTGTACACTGCC 100 nM 2614-2637 Probe*** TGTGACYGC(SEQ ID NO: 8) *The concentration of each oligonucleotide in the finalreaction mixture is provided. **Genomic locations are provided based onthe reference sequence Chikungunya virus strain S27-African prototype(Genbank: AF369024.2). ***The 5′ fluor and 3′ quencher on the CHIKVprobe were Cal Fluor 610 and BHQ-2, respectively.

TABLE 2 Comparison of DENV detection in the pan-DENV-CHIKV rRT-PCR withdetection in the hemi-nested DENY RT-PCR. Hemi-nested DENV RT-PCRPositive Negative Total pan-DENV- Positive 74 7* 81 CHIKV Negative 1 100101 rRT-PCR Total 75 107 182 *Five of seven patients also test edpositive for DENY by IgM MAC-ELISA or Inhibition ELISA.

TABLE 3 Comparisn of CHIK V detection in the pan-DENV-CHIKV rRT-PCR withdetection in the reference CHIKV rRT-PCR. Comparator CHIKV rRT-PCRPositive Negative Total pan-DENV- Positive 57 0 57 CHIKV Negative 0 2424 rRT-PCR Total 57 24 81

TABLE 4 Primer sequences for the dengue  multiplex rRT-PCR. NamePrimer Sequence (5′→3′) Dengue 1-2-3 Forward CAGATCTCTGATGAACAACCAACG(SEQ ID NO: 9) Dengue 2 Forward C→T CAGATCTCTGATGAATAACCAACG(SEQ ID NO: 10) Dengue 3 Forward C→T CAGATTTCTGATGAACAACCAACG(SEQ ID NO: 11) Dengue 4 Forward GATCTCTGGAAAAATGAAC (SEQ ID NO: 12)Dengue 1, 3 Reverse TTTGAGAATCTCTTCGCCAAC (SEQ ID NO: 13)Dengue 2 Reverse AGTTGACACGCGGTTTCTCT (SEQ ID NO: 14)Dengue 2 Reverse A→G AGTCGACACGCGGTTTCTCT (SEQ ID NO: 15)Dengue 4 Reverse AGAATCTCTTCACCAACC (SEQ ID NO: 16)

TABLE 5 Probe sequences for the dengue multiplex rRT-PCR. Probe SequenceChannel  5′ Fluor (5′→3′) 3′ Quencher Green FAM CGCGATCGCGTTTCAGCATBHQ-1 ATTGAAAGACGGATCGCG (SEQ ID NO: 17) Yellow CAL FluorCGCGATCGCGTTTCAGCAT BHQ-1 Orange 560 ATTGAAAGGCGGATCGCG (SEQ ID NO: 18)Orange CAL Fluor CGCGATCCACGCGTTTCAG BHQ-2 Red 610 CATATTGATAGGATCGCG(SEQ ID NO: 19) Red Quasar CGCGATCTTTCAGCATATT  BHQ-2 Blue 670GAAAGGTGGTCGATCGCG (SEQ ID NO: 20)Probes are listed by the channel in which signal is detected on the Rotor-Gene Q instrument.Underlined probe segments designate sequences complimentary to the dengue consensus; segments on the 5′ and 3′ ends of the probe comprise the beaconstem. Beacon Name 5′ Fluor Sequence 3′Quencher Den1 FAMCGCGATCTTCAGCATATT  BHQ-1 Alternate GAAAGACGGTCGGATCG CG (SEQ ID NO: 21)Taqman Name 5′ Fluor Sequence 3′Quencher DENV1 BHQ+ FAMCTCGCGCGTTTCAGCATAT BHQplus (SEQ ID NO: 22) DENV2 BHQ+ FAMCTCTCGCGTTTCAGCATAT BHQplus (SEQ ID NO: 23) DENV2 Alt BHQ+  FAMCTCTCACGTTTCAGCATA BHQplus TTG (SEQ ID NO: 24) DENV3 BHQ+ FAMCTCACGCGTTTCAGCATAT BHQplus (SEQ ID NO: 25)

Example 2 Real-time RT-PCR for the Detection and Quantitation of ZikaVirus with the Capability for Multiplexed Detection of Dengue andChikungunya Viruses

Introduction

Here, we describe the design and analytical and clinical evaluation of anew rRT-PCR assay for Zika virus (ZIKV). This assay can be used alone orcombined in a multiplex assay for DENV and CHIKV (hereinafter referredto as the DCZ assay).

ZIKV is a mosquito-borne flavivirus that was first identified in Ugandain 1947. Prior to 2007, very few cases of human infection with ZIKV hadbeen identified and all had occurred in Africa or Asia. In 2007, anoutbreak of ZIKV occurred on Yap Island, and in May 2015, ZIKV wasidentified in Brazil. This was the first documented transmission of ZIKVin the Western Hemisphere, and the virus has now spread to othercountries in South America as well as Mexico, Central America, andislands in the Caribbean. Clinically, ZIKV cannot be reliablydistinguished from infections with two other common arboviruses in thisregion, dengue virus (DENV) and chikungunya virus (CHIKV). Two real-timereverse-transcriptase PCRs (rRT-PCRs) for the detection of ZIKV havebeen published, though both assays are used solely in monoplex reactions(i.e. only for ZIKV). The purpose of the current project was to developa new rRT-PCR for the detection and quantitation of ZIKV from patientsamples as well as to optimize a multiplex, single-reaction rRT-PCR forthe detection and differentiation of DENV, CHIKV, and ZIKV.

From an alignment of available ZIKV complete or near-complete genomesequences in GenBank, a new ZIKV rRT-PCR was designed for use inmultiplex with assays for pan-DENV detection and CHIKV detection.Analytical evaluation of the ZIKV assay was performed as part of themultiplex reaction for DENV, CHIKV and ZIKV (referred to as the DCZassay) in accordance with published recommendations. The ZIKV rRT-PCRhad a dynamic range extending from 8.0 to 1.0 log₁₀ copies/μL and alower limit of 95% detection of 7.8 copies/μL. The DCZ assay was used totest 227 serum samples from patients with an acute febrile illness inNicaragua. 177 patients tested positive using the DCZ assay, including30 patients with DENV, 110 with CHIKV, 36 with DENV-CHIKV co-infections,and one patient with a ZIKV-CHIKV co-infection.

We have developed a new rRT-PCR for ZIKV and demonstrated that this testhas a wide linear range and provides sensitive detection of viral RNA.Furthermore, the performance of this assay was evaluated in thesingle-reaction, multiplex DCZ assay. This assay provides sensitivedetection of three important human arboviruses and has the potential toimprove virus detection while decreasing testing costs and streamliningmolecular workflow.

Methods

rRT-PCR Design and Performance. To design the ZIKV rRT-PCR, all completeor nearly-complete (≥10,000 kb) ZIKV genome sequences available inGenBank (n=21; accessed 28 Mar. 2014) were aligned using MegAlignsoftware (DNAStar). A consensus sequence was generated that identifiedbases conserved across ≥95% of available sequences. Primers and probeswere designed from the consensus sequence using Primer3 software. Fourtarget regions were identified; primers and probes were designed andevaluated for each region. ZIKV primers and probes were tested in silicousing BLASTn to search the National Center for Biotechnology Information(NCBI) nucleotide database. The final primer-probe set was selected forimproved analytical sensitivity and maintained in silico specificity. Todetermine optimal concentrations in the final reaction, the ZIKV primersand probe were tested, in multiplex with the pan-DENV and CHIKVrRT-PCRs, at each combination of 100, 200, and 400 nM primer and 100,200, and 400 nM probe. The concentration of DENV primers and probes inthe final reaction were maintained from the pan-DENV-CHIKV rRT-PCR(Waggoner et al. (2016) J. Clin. Virol. 78:57-61). The CHIKV primers andprobe (Table 2) were used at 300 nM and 100 nM, respectively, in thefinal DCZ reaction.

The DCZ assay and ZIKV rRT-PCR were performed in a total volume of 25 μLusing the SuperScript III Platinum One-Step qRT-PCR kit (LifeTechnologies) and 5 μL of nucleic acid eluate. ZIKV primer and probeconcentrations in the final reaction mixture are listed in Table 6.Evaluation was performed on a Rotor-Gene Q instrument at StanfordUniversity (RGQ, Qiagen) and an ABI7500 instrument (Life Technologies)at the National Virology Laboratory in Managua, Nicaragua. Cyclingconditions were the following: 52° C. for 15 minutes; 94° C. for 2minutes; 45 cycles of 94° C. for 15 seconds, 55° C. for 40 seconds, and68° C. for 20 seconds. Each run was performed with a negative control(no template) and a positive control for ZIKV. The DCZ assay wasperformed with controls for DENV, CHIKV and ZIKV. Signal was acquired at55° C., and analysis was performed on the linear scale. Thresholds wereset manually on each instrument and used for analysis of each run. Forboth targets, any exponential curve crossing this threshold wasconsidered positive.

Control Nucleic Acids and Reference Material. Quantitated,positive-sense, single-stranded DNA (ssDNA) oligonucleotides containingthe target sequence for ZIKV, each DENV serotype, and CHIKV weresynthesized (Eurofins MWG Operon) and used in the analyticalcharacterization of the ZIKV rRT-PCR and DCZ assays. The specificity ofthe ZIKV rRT-PCR, as a component of the DCZ assay, was evaluated bytesting genomic RNA from the following viruses: DENV-1 Hawaii 1944,DENV-2 New Guinea C strain, DENV-3 strain H87, and DENV-4 strain H241,CHIKV (strain R80422a provided by the CDC Division of Vector BorneDiseases), West Nile (NY 1999 strain), and hepatitis C virus (genotype1).

Analytical Characterization. The analytical evaluation of the ZIKVrRT-PCR was performed as part of the DCZ assay according to publishedrecommendations (Burd (2010) Clin. Microbiol. Rev. 23:550-576) and aspreviously described (Waggoner et al. (2013) J. Clin. Microbiol.51:2172-2181; Waggoner et al. (2014) J. Clin. Microbiol. 52:2011-18).Briefly, for ZIKV, each DENV serotype, and CHIKV, linearity studies wereperformed using serial 10-fold dilutions of quantified ssDNA. Fourreplicates of each dilution from 8.0 log₁₀ copies/μL to 1 copy/μL weretested on a single run. The linear range was established by fitting abest-fit line to the data by regression analysis and included the rangewhere the R² value was ≥0.99. To establish the lower limit of 95%detection (95% LLOD), ten replicates of five 2-fold dilutions from 50 to3.12 copies/μL were tested on a single run. The 95% LLOD was thencalculated using probit analysis.

Clinical Samples and Nucleic Acid Extraction. Pre-collected andde-identified serum samples from 227 Nicaraguan patients with suspecteddengue, chikungunya, or Zika were used for the clinical evaluation ofthe DCZ assay. Samples were collected during 2015 as part of theNicaraguan National Surveillance System and two ongoing studies: theNicaraguan Pediatric Dengue Cohort Study and a hospital-based denguestudy, both based in Managua. Study design and methods for both of thesestudies have been described (Hammond et al. (2005) Am. J. Trop. Med.Hyg. 73:1063; Harris et al. (2000) Am. J. Trop. Med. Hyg. 63:5-11; Kuanet al. (2009) Am. J. Epidemiol. 170:120-129). RNA extractions wereperformed using the QIAamp Viral RNA Mini Kit (Qiagen) with 140 μL ofserum using and an elution volume of 60 μL.

Statistics. Kappa statistics were calculated with GraphPad software(GraphPad) and used to compare RT-PCR results. CT values from DENV- andCHIKV-positive clinical samples were compared by unpaired t test(GraphPad). Probit analysis was performed using SPSS (IBM) to determinethe 95% LLOD for the ZIKV rRT-PCR.

Results

Analytical Evaluation. Using serial 10-fold dilutions of ssDNA, thelinear range of the pan-DENV-CHIKV rRT-PCR for ZIKV extended from 8.0 to1.0 log₁₀ copies/μL. The 95% LLOD was calculated to be 7.8 copies/μL(5.7-27.2, 95% CI). Amplification of ZIKV, DENV and CHIKV controls wasdetected in the appropriate channels without cross-reaction. Noamplification was observed when the DCZ assay was performed usinggenomic RNA from the aforementioned viruses (see Material and Methods).

Clinical Samples. Sera from 227 Nicaraguan patients with suspecteddengue, chikungunya, or Zika were tested using the DCZ assay. 177patients (78.0%) tested positive for one or more viruses: 110 for CHIKVmono-infection, 30 for DENV mono-infection, 36 for DENV-CHIKVco-infection, and 1 patient for CHIKV-ZIKV co-infection. The remaining50 patients (22.0%) were negative using the DCZ assay.

Discussion

We describe the development a new rRT-PCR for ZIKV and the analyticaland clinical evaluation of this rRT-PCR as a component of the DCZ assay.The DCZ multiplex assay demonstrated excellent analytical sensitive, awide dynamic range, and provided an etiologic diagnosis for ahigh-percentage of patients with an acute febrile illness and suspectedarbovirus infection. The DCZ assay also provides all of this a single,real-time reaction. Multiplex rRT-PCRs for the detection of DENV, CHIKV,and ZIKV have not been reported to date. Recommended testing protocolsfor these viruses often involve the performance of separate moleculartests for each virus, which can amount to six separate reactions intotal. The goal of our study was, therefore, to design an rRT-PCR forZIKV that would be compatible with previously designed assays forpan-DENV and CHIKV detection (Waggoner et al. (2016) J. Clin. Virol.78:57-61). The resulting DCZ assay was then evaluated using clinicalsamples collected in Nicaragua, which is experiencing the transmissionof DENV and CHIKV currently.

In conclusion, we report the development and evaluation of a new ZIKVrRT-PCR and the DCZ assay. This assay demonstrated very good analyticaland clinical performance. The single-reaction, multiplex format of theDCZ assay, combined with sensitive detection of both viruses, has thepotential to improve detection while decreasing test costs andstreamlining molecular workflow.

TABLE 6 ZIKV primer and probe sequences. Concentra- Loca- NameSequence (5′→3′) tion^(a) tion^(b) ZIKV CAGCTGGCAT 300 nM 7332-7352Forward CATGAAGAAYC (SEQ ID NO: 27) ZIKV CACTTGTCCCA 300 nM 7411-7432Reverse TCTTCTTCTCC 1 (SEQ ID NO: 28) ZIKV CACCTGTCCCAT 300 nM 7411-7432Reverse CTTTTTCTCC 2 (SEQ ID NO: 29) ZIKV CYGTTGTGGAT 100 nM 7355-7373Probe GGAATAGTGG (SEQ ID NO: 30) ^(a)The concentration of eacholigonucleotide in the final reaction mixture is provided. ^(b)Genomiclocations are provided based on the reference sequence Zika virusstrain: MR766-NIID (Genbank: LC002520.1).

TABLE 7 CHIKV primer and probe sequences for the pan-ZIKV-CHIKV rRT-PCR.Concentra- Loca- Name Sequence (5′→3′) tion^(*) tion^(**) CHIKVCATCTGCACYC 300 nM 2578-2598 Forward AAGTGTACCA (SEQ ID NO: 6) CHIKVGCGCATTTTGC 300 nM 2654-2674 Reverse CTTCGTAATG (SEQ ID NO: 7) CHIKVGCGGTGTACAC 100 nM 2614-2637 Probe*** TGCCTGTGACYGC (SEQ ID NO: 8) *Theconcentration of each oligonucleotide in the final reaction mixture isprovided. **Genomic locations are provided based on the referencesequence Chikungunya virus strain S27-African prototype (Genbank:AF369024.2). ***The 5′ fluor and 3′ quencher on the CHIKV probe were CalFluor 610 and BHQ-2, respectively.

Thus, oligonucleotide reagents, including primers and probes, as well asmethods of using the reagents for detection of chikungunya, Zika, anddengue viruses are described. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined herein.

1. A composition for detecting chikungunya virus in a biological sampleusing a nucleic acid amplification assay, the composition comprising atleast one set of oligonucleotide primers comprising a forward primer anda reverse primer capable of amplifying at least a portion of achikungunya virus genome, wherein said primers are not more than 40nucleotides in length, wherein said set of primers is selected from thegroup consisting of: a) a forward primer comprising the nucleotidesequence of SEQ ID NO:6 and a reverse primer comprising the sequence ofSEQ ID NO:7; b) a forward primer comprising at least 10 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:6 and a reverseprimer comprising at least 10 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:7; c) a forward primer comprising a nucleotidesequence having at least 95% identity to the sequence of SEQ ID NO:6 anda reverse primer comprising a nucleotide sequence having at least 95%identity to the sequence of SEQ ID NO:7, wherein the primer is capableof hybridizing to and amplifying chikungunya virus nucleic acids in thenucleic acid amplification assay; d) a forward primer and a reverseprimer comprising at least one nucleotide sequence that differs from thecorresponding nucleotide sequence of the forward primer or reverseprimer of the primer set of (a) in that the primer has up to threenucleotide changes compared to the corresponding sequence, wherein theprimer is capable of hybridizing to and amplifying chikungunya virusnucleic acids in the nucleic acid amplification assay; and e) a forwardprimer and a reverse primer comprising nucleotide sequences that arecomplements of the corresponding nucleotide sequences of the forwardprimer and reverse primer of a primer set selected from the groupconsisting of (a)-(d).
 2. The composition of claim 1, further comprisingat least one detectably labeled oligonucleotide probe sufficientlycomplementary to and capable of hybridizing with a chikungunya virus RNAor an amplicon thereof.
 3. The composition of claim 2, wherein the probeis selected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:8; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:8, wherein theprobe is capable of hybridizing to and detecting the chikungunya virusRNA or an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:8 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the chikungunya virus RNA or an amplicon thereof.
 4. Thecomposition of claim 2, wherein the detectably labeled probe comprises afluorophore.
 5. The composition of claim 4, wherein the detectablylabeled probe comprises a 5′- fluorophore and a 3′-quencher. 6-74.(canceled)
 75. A composition for detecting Zika virus in a biologicalsample using a nucleic acid amplification assay, the compositioncomprising at least one set of oligonucleotide primers comprising aforward primer and a reverse primer capable of amplifying at least aportion of a Zika virus genome, wherein said primers are not more than40 nucleotides in length, wherein said set of primers is selected fromthe group consisting of: a) a forward primer comprising the nucleotidesequence of SEQ ID NO:27 and a reverse primer comprising the sequence ofSEQ ID NO:28; b) a forward primer comprising the nucleotide sequence ofSEQ ID NO:27 and a reverse primer comprising the sequence of SEQ IDNO:29; c) a forward primer comprising the nucleotide sequence of SEQ IDNO:27, a first reverse primer comprising the sequence of SEQ ID NO:28,and a second reverse primer comprising the sequence of SEQ ID NO:29; d)a forward primer comprising at least 10 contiguous nucleotides of thenucleotide sequence of SEQ ID NO:27 and at least one reverse primercomprising at least 10 contiguous nucleotides of a nucleotide sequenceselected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29; e)a forward primer comprising a nucleotide sequence having at least 95%identity to the sequence of SEQ ID NO:27 and at least one reverse primercomprising a nucleotide sequence having at least 95% identity to asequence selected from the group consisting of SEQ ID NO:28 and SEQ IDNO:29, wherein the primer is capable of hybridizing to and amplifyingZika virus nucleic acids in the nucleic acid amplification assay; f) aforward primer and a reverse primer comprising at least one nucleotidesequence that differs from the corresponding nucleotide sequence of theforward primer or reverse primer of the primer set of (a) or (b) in thatthe primer has up to three nucleotide changes compared to thecorresponding sequence, wherein the primer is capable of hybridizing toand amplifying Zika virus nucleic acids in the nucleic acidamplification assay; and g) a forward primer and a reverse primercomprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(f).76. The composition of claim 75, further comprising at least onedetectably labeled oligonucleotide probe sufficiently complementary toand capable of hybridizing with a Zika virus RNA or an amplicon thereof.77. The composition of claim 76, wherein the probe is selected from thegroup consisting of: a) a probe comprising the sequence of SEQ ID NO:30;b) a probe comprising a nucleotide sequence having at least 95% identityto the sequence of SEQ ID NO:30, wherein the probe is capable ofhybridizing to and detecting the Zika virus RNA or an amplicon thereof;and c) a probe that differs from the corresponding nucleotide sequenceof SEQ ID NO:30 by up to three nucleotide changes, wherein the probe iscapable of hybridizing to and detecting the Zika virus RNA or anamplicon thereof.
 78. The composition of claim 76, wherein thedetectably labeled probe comprises a fluorophore.
 79. The composition ofclaim 78, wherein the detectably labeled probe comprises a 5′-fluorophore and a 3′-quencher. 80-85. (canceled)
 86. A method fordetecting Zika virus, the method comprising: a) contacting nucleic acidsof a biological sample suspected of containing Zika virus with thecomposition of claim 75; b) amplifying at least a portion of a Zikavirus RNA, if present; and c) detecting the presence of the amplifiednucleic acids using at least one detectably labeled oligonucleotideprobe sufficiently complementary to and capable of hybridizing with theZika virus RNA or amplicon thereof, if present, as an indication of thepresence or absence of Zika virus in the sample.
 87. The method of claim86, wherein the probe is selected from the group consisting of: a) aprobe comprising the sequence of SEQ ID NO:30; b) a probe comprising anucleotide sequence having at least 95% identity to the sequence of SEQID NO:30, wherein the probe is capable of hybridizing to and detectingthe Zika virus RNA or an amplicon thereof; and c) a probe that differsfrom the corresponding nucleotide sequence of SEQ ID NO:30 by up tothree nucleotide changes, wherein the probe is capable of hybridizing toand detecting the Zika virus RNA or an amplicon thereof.
 88. The methodof claim 86, wherein the detectably labeled probe comprises afluorophore.
 89. The method of claim 88, wherein the detectably labeledprobe comprises a 5′- fluorophore and a 3′-quencher.
 90. The method ofclaim 89, wherein the 5′-fluorophore is a fluorescein or rhodaminederivative. 91-124. (canceled)
 125. A kit for detecting Zika virus in abiological sample, the kit comprising: written instructions foridentifying the presence of Zika virus; and at least one set of primerscomprising a forward primer and a reverse primer capable of amplifyingat least a portion of a Zika virus genome, wherein said primers are notmore than about 40 nucleotides in length, wherein said set of primers isselected from the group consisting of: a) a forward primer comprisingthe nucleotide sequence of SEQ ID NO:27 and a reverse primer comprisingthe sequence of SEQ ID NO:28; b) a forward primer comprising thenucleotide sequence of SEQ ID NO:27 and a reverse primer comprising thesequence of SEQ ID NO:29; c) a forward primer comprising the nucleotidesequence of SEQ ID NO:27, a reverse primer comprising the sequence ofSEQ ID NO:28, and a reverse primer comprising the sequence of SEQ IDNO:29; d) a forward primer comprising at least 10 contiguous nucleotidesof the nucleotide sequence of SEQ ID NO:27 and at least one reverseprimer comprising at least 10 contiguous nucleotides of a nucleotidesequence selected from the group consisting of SEQ ID NO:28 and SEQ IDNO:29; e) a forward primer comprising a nucleotide sequence having atleast 95% identity to the sequence of SEQ ID NO:27 and at least onereverse primer comprising a nucleotide sequence having at least 95%identity to a sequence selected from the group consisting of SEQ IDNO:28 and SEQ ID NO:29, wherein the primer is capable of hybridizing toand amplifying Zika virus nucleic acids in the nucleic acidamplification assay; f) a forward primer and a reverse primer comprisingat least one nucleotide sequence that differs from the correspondingnucleotide sequence of the forward primer or reverse primer of theprimer set of (a) or (b) in that the primer has up to three nucleotidechanges compared to the corresponding sequence, wherein the primer iscapable of hybridizing to and amplifying Zika virus nucleic acids in thenucleic acid amplification assay; and g) a forward primer and a reverseprimer comprising nucleotide sequences that are complements of thecorresponding nucleotide sequences of the forward primer and reverseprimer of a primer set selected from the group consisting of (a)-(f).126. The kit of claim 125, further comprising at least one probe fordetecting Zika virus in a biological sample, wherein the probe isselected from the group consisting of: a) a probe comprising thesequence of SEQ ID NO:30; b) a probe comprising a nucleotide sequencehaving at least 95% identity to the sequence of SEQ ID NO:30, whereinthe probe is capable of hybridizing to and detecting the Zika virus RNAor an amplicon thereof; and c) a probe that differs from thecorresponding nucleotide sequence of SEQ ID NO:30 by up to threenucleotide changes, wherein the probe is capable of hybridizing to anddetecting the Zika virus RNA or an amplicon thereof.
 127. The kit ofclaim 126, wherein the detectably labeled probe comprises a fluorophore.128. The kit of claim 127, wherein the detectably labeled probecomprises a 5′-fluorophore and a 3′-quencher. 129-140. (canceled)